End-Modified Poly(beta-amino esters) and Uses Thereof

ABSTRACT

Poly(beta-amino esters) are end-modified to form materials useful in the medical as well as non-medical field. An amine-terminated poly(beta-amino ester) is reacted with an electrophile, or an acrylate-terminated poly(beta-amino ester) is reacted with a nucleophile. The inventive end-modified polymers may be used in any field where polymers have been found useful including the drug delivery arts. The end-modified polymers are particularly useful in delivery nucleic acids such as DNA or RNA. The invention also provides compositions including the inventive end-modified polymers, methods of preparing the inventive polymers, and method of using the inventive polymers.

RELATED APPLICATIONS

The present application claims priority under 35 U.S.C. § 119(e) to U.S.provisional patent application, U.S. Ser. No. 60/832,517, filed Jul. 21,2006, which is incorporated herein by reference.

The present application is also related but does not claim priority toU.S. patent applications, U.S. Ser. No. 11/099,886, filed Apr. 6, 2005;U.S. Ser. No. 10/446,444, filed May 28, 2003; U.S. Ser. No. 09/969,431,filed Oct. 2, 2001; U.S. Ser. No. 60/305,337, filed Jul. 13, 2001; andU.S. Ser. No. 60/239,330, filed Oct. 10, 2000; each of which isincorporated herein by reference.

GOVERNMENT SUPPORT

This invention was made with United States Government support underCooperative Agreement #ECC9843342 to the MIT Biotechnology ProcessEngineering Center by the National Science Foundation, under contractGM26698 and NRSA Fellowship # 1 F32 GM20227-01 by the NationalInstitutes of Health, and under Cooperative Agreement DAMD 17-99-2-9-001to the Center for Innovative Minimally Invasive Therapy by theDepartment of the Army. The United States Government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

The treatment of human diseases through the application ofnucleotide-based drugs such as DNA and RNA has the potential torevolutionize the field of medicine (Anderson Nature 392(Suppl.):25-30,1996; Friedman Nature Med. 2:144-147, 1996; Crystal Science 270:404-410,1995; Mulligan Science 260:926-932, 1993; each of which is incorporatedherein by reference). Thus far, the use of modified viruses as genetransfer vectors has generally represented the most clinicallysuccessful approach to gene therapy. While viral vectors are currentlythe most efficient gene transfer agents, concerns surrounding theoverall safety of viral vectors, which include the potential forunsolicited immune responses, have resulted in parallel efforts todevelop non-viral alternatives (for leading references, see: Luo et al.Nat. Biotechnol. 18:33-37, 2000; Behr Acc. Chem. Res. 26:274-278, 1993;each of which is incorporated herein by reference). Current alternativesto viral vectors include polymeric delivery systems (Zauner et al. Adv.Drug Del. Rev. 30:97-113, 1998; Kabanov et al. Bioconjugate Chem.6:7-20, 1995; each of which is incorporated herein by reference),liposomal formulations (Miller Angew. Chem. Int. Ed. 37:1768-1785, 1998;Hope et al. Molecular Membrane Technology 15:1-14, 1998; Deshmukh et al.New J. Chem. 21:113-124, 1997; each of which is incorporated herein byreference), and “naked” DNA injection protocols (Sanford TrendsBiotechnol. 6:288-302, 1988; incorporated herein by reference). Whilethese strategies have yet to achieve the clinical effectiveness of viralvectors, the potential safety, processing, and economic benefits offeredby these methods (Anderson Nature 392(Suppl.):25-30, 1996; incorporatedherein by reference) have ignited interest in the continued developmentof non-viral approaches to gene therapy (Boussif et al Proc. Natl. Acad.Sci. USA 92:7297-7301, 1995; Putnam et al Macromolecules 32:3658-3662,1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999; Gonzalez et al.Bioconjugate Chem. 10:1068-1074, 1999; Kukowska-Latallo et al. Proc.Natl. Acad. Sci. USA 93:4897-4902, 1996; Tang et al. Bioconjugate Chem.7:703-714, 1996; Haensler et al. Bioconjugate Chem. 4:372-379, 1993;each of which is incorporated herein by reference).

Cationic polymers have been widely used as transfection vectors due tothe facility with which they condense and protect negatively chargedstrands of DNA. Amine-containing polymers such as poly(lysine) (Zauneret al. Adv. Drug Del. Rev. 30:97-113, 1998; Kabanov et al. BioconjugateChem. 6:7-20, 1995; each of which is incorporated herein by reference),poly(ethylene imine) (PEI) (Boussif et al Proc. Natl. Acad. Sci. USA92:7297-7301, 1995; incorporated herein by reference), andpoly(amidoamine) dendrimers (Kukowska-Latallo et al. Proc. Natl. Acad.Sci. USA 93:4897-4902, 1996; Tang et al. Bioconjugate Chem. 7:703-714,1996; Haensler et al. Bioconjugate Chem. 4:372-379, 1993; each of whichis incorporated herein by reference) are positively-charged atphysiological pH, form ion pairs with nucleic acids, and mediatetransfection in a variety of cell lines. Despite their common use,however, cationic polymers such as poly(lysine) and PEI can besignificantly cytotoxic (Zauner et al. Adv. Drug Del. Rev. 30:97-113,1998; Deshmukh et al. New J. Chem. 21:113-124, 1997; Choksakulnimitr etal. Controlled Release 34:233-241, 1995; Brazeau et al. Pharm. Res.15:680-684, 1998; each of which is incorporated herein by reference). Asa result, the choice of cationic polymer for a gene transfer applicationgenerally requires a trade-off between transfection efficiency andshort- and long-term cytotoxicity. Additionally, the long-termbiocompatibility of these polymers remains an important issue for use intherapeutic applications in vivo, since several of these polymers arenot readily biodegradable (Uhrich Trends Polym. Sci. 5:388-393, 1997;Roberts et al. J. Biomed. Mater. Res. 30:53-65, 1996; each of which isincorporated herein by reference).

In order to develop safe alternatives to existing polymeric vectors andother functionalized biomaterials, degradable polyesters bearingcationic side chains have been developed (Putnam et al. Macromolecules32:3658-3662, 1999; Barrera et al. J. Am. Chem. Soc. 115:11010-11011,1993; Kwon et al. Macromolecules 22:3250-3255, 1989; Lim et al. J. Am.Chem. Soc. 121:5633-5639, 1999; Zhou et al. Macromolecules 23:3399-3406,1990; each of which is incorporated herein by reference). Examples ofthese polyesters include poly(L-lactide-co-L-lysine) (Barrera et al. J.Am. Chem. Soc. 115:11010-11011, 1993; incorporated herein by reference),poly(serine ester) (Zhou et al. Macromolecules 23:3399-3406, 1990; eachof which is incorporated herein by reference), poly(4-hydroxy-L-prolineester) (Putnam et al. Macromolecules 32:3658-3662, 1999.; Lim et al. J.Am. Chem. Soc. 121:5633-5639, 1999; each of which is incorporated hereinby reference), and more recently, poly[α-(4-aminobutyl)-L-glycolicacid]. Poly(4-hydroxy-L-proline ester) andpoly[α-(4-aminobutyl)-L-glycolic acid] were recently demonstrated tocondense plasmid DNA through electrostatic interactions, and to mediategene transfer (Putnam et al. Macromolecules 32:3658-3662, 1999; Lim etal. J. Am. Chem. Soc. 121:5633-5639, 1999; each of which is incorporatedherein by reference). Importantly, these new polymers are significantlyless toxic than poly(lysine) and PEI, and they degrade into non-toxicmetabolites. It is clear from these investigations that the rationaldesign of amine-containing polyesters can be a productive route to thedevelopment of safe, effective transfection vectors. Unfortunately,however, present syntheses of these polymers require either theindependent preparation of specialized monomers (Barrera et al. J. Am.Chem. Soc. 115:11010-11011, 1993; incorporated herein by reference), orthe use of stoichiometric amounts of expensive coupling reagents (Putnamet al. Macromolecules 32:3658-3662, 1999; incorporated herein byreference). Additionally, the amine functionalities in the monomers mustbe protected prior to polymerization (Putnam et al. Macromolecules32:3658-3662, 1999; Lim et al. J. Am. Chem. Soc. 121:5633-5639, 1999;Gonzalez et al Bioconjugate Chem. 10:1068-1074, 1999; Barrera et al. J.Am. Chem. Soc. 115:11010-11011, 1993; Kwon et al. Macromolecules22:3250-3255, 1989; each of which is incorporated herein by reference),necessitating additional post-polymerization deprotection steps beforethe polymers can be used as transfection agents.

There exists a continuing need for non-toxic, biodegradable,biocompatible polymers that can be used to transfect nucleic acids andthat are easily prepared efficiently and economically. Such polymerswould have several uses, including the delivery of nucleic acids in genetherapy as well as in the packaging and/or delivery of diagnostic,therapeutic, and prophylactic agents.

SUMMARY OF THE INVENTION

The present invention provides novel end-modified poly(beta-aminoesters) useful in a variety of medical applications including drugdelivery, tissue engineering, and biomaterials and non-medicalapplications including coatings, plastics, paints, and films. In certainembodiments, the inventive end-modified poly(beta-amino esters) areprepared by the addition of a nucleophilic reagent (e.g., an amine) toan acrylate-terminated poly(beta-amino ester). In other embodiments, theinventive end-modified poly(beta-amino esters) are prepared by theaddition of an electrophilic reagent (e.g., acrylate, acrylamide) to anacrylate-terminated poly(beta-amino ester). Any acrylate-terminatedpoly(beta-amino ester) or nucleophilic reagent may be used to prepare anend-modified poly(beta-amino ester). In addition to poly(beta-aminoesters), poly(beta-amino amides) as well as other polymers with reactiveend moieties may be end-modified. The resulting end-modified polymersare useful in drug delivery, particularly in the delivery ofpolynucleotides. The invention also provides complexes of the inventiveend-modified polymers with polynucleotides, drug delivery devices (e.g.,microparticles, nanoparticles) including the inventive polymers, methodsof preparing end-modified poly(beta-amino esters), and methods of usingthe inventive end-modified polymers.

In one aspect, the invention provides end-terminated poly(beta-aminoesters). The inventive polymers are generally of one of the formulae:

wherein A and B are linkers which may be any substituted orunsubstituted, branched or unbranched, cyclic or acyclic aliphatic orheteroaliphatic moiety; or substituted or unsubstituted aryl orheteroaryl moieties;

each of R₁, R₂, R₃, and R₄ are independently a hydrogen; halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and

X is O, S, NH, or NR_(X), wherein R_(X) is halogen; branched orunbranched, substituted or unsubstituted, cyclic or acyclic aliphatic;branched or unbranched, substituted or unsubstituted, cyclic or acyclicheteroaliphatic; branched or unbranched, substituted or unsubstituted,cyclic or acyclic acyl; substituted or unsubstituted aryl; orsubstituted or unsubstituted heteroaryl. In certain embodiments, theterminal groups of the end-modified polymers are the same. In otherembodiments, the terminal groups are different. Any of thepoly(beta-amino esters) described in U.S. patent applications U.S. Ser.No. 09/969,431 and U.S. Ser. No. 10/446,444, each of which isincorporated herein by reference, could be used to prepare anend-modified polymer. The molecular weights of the inventive polymersmay range from 1000 g/mol to 20,000 g/mol, preferably from 5,000 g/molto 15,000 g/mol.

In another aspect, the invention provides end-terminated poly(beta-aminoamides). The inventive polymers are generally of one of the formulae:

wherein A and B are linkers which may be any substituted orunsubstituted, branched or unbranched, cyclic or acyclic aliphatic orheteroaliphatic moiety; or substituted or unsubstituted aryl orheteroaryl moieties;

each R′ is independently a hydrogen; branched or unbranched, substitutedor unsubstituted, cyclic or acyclic aliphatic; branched or unbranched,substituted or unsubstituted, cyclic or acyclic heteroaliphatic;branched or unbranched, substituted or unsubstituted, cyclic or acyclicacyl; substituted or unsubstituted aryl; or substituted or unsubstitutedheteroaryl;

each of R₁, R₂, R₃, and R₄ are independently a hydrogen; halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and

each X is independently O, S, NH, or NR_(X), wherein R_(X) is halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and salts thereof. Incertain embodiments, the terminal groups of the end-modified polymersare the same. In other embodiments, the terminal groups are different.In certain embodiments, all R′ are hydrogen. In other embodiments, allR′ are C₁-C₆ alkyl. The molecular weights of the inventive polymers mayrange from 1000 g/mol to 20,000 g/mol, preferably from 5,000 g/mol to15,000 g/mol. In certain embodiments, a salt of the inventiveend-modified polymers is used, for example, cationic salts such assodium, magnesium, potassium, zinc, calcium, etc., or anionic salts suchchloride, bromide, iodide, sulfate, phosphate, etc.

The present invention also provides end-modified polymers wherein theends of the polymers are serially modified. For example, in certainembodiments, an acrylate-terminated poly(beta-amino ester) orpoly(beta-amino amide) is reacted with a nucleophile which results inanother reactive moiety (e.g., an amino group, hydroxyl group, thiolgroup) being placed at the end of the polymer. This reactive moiety issubsequently modified. For example, a terminal amino group may besubsequently modified by the addition of an electrophile (e.g., anacrylate, acrylamide, alkyl halide, etc.). To give but another example,in certain embodiments, an amine-terminated poly(beta-amino ester) orpoly(beta-amino amide) is reacted with an electrophile which results inanother reactive moiety (e.g., an amino group, hydroxyl group, thiolgroup) being placed at the end of the polymer. This process of seriallymodifying the end of a polymer can be continued for any number ofrounds. In certain embodiments, the process is continued for at least 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more rounds. The desired polymer mayoptionally be purified after each round of end-modification.

The present invention also provides methods of preparing the inventiveend-modified poly(beta-amino esters) and poly(beta-amino esters). Incertain embodiments, the methods begins by starting with anacylate-terminated polymer or by preparing such a polymer from abisacrylate and an amine, or a bisacrylamide and an amine. Theacrylate-terminated polymer is reacted with a nucleophile underconditions suitable for the nucleophile to add to the terminal acrylatemoieties of the polymer. In certain other embodiments, the methodsbegins by starting with an amine-terminated polymer or by preparing sucha polymer from a bisacrylate and an amine, or a bisacrylamide and anamine. The amine-terminated polymer is reacted with an electrophileunder conditions suitable for the nucleophile to add to the terminalamine moieties of the polymer. The resulting end-modified polymers maythen be optionally purified or characterized. The inventive polymer mayfind use in drug delivery or other biomedical applications. Theinventive polymers may also be used in the myriad of ways other polymersare used. For example, the inventive polymers may be used inmanufacturing materials, coatings, nanodevices, etc.

In certain aspects of the invention, the inventive polymers are used toencapsulate therapeutic, diagnostic, and/or prophylactic agentsincluding polynucleotides, peptides, proteins, cells, biomolecules,small molecules, etc. For example, the end-modified polymers may be usedto form particles, microparticles, nanoparticles, or other drug deliverydevices. The end-modified polymers terminated with an amine or othergroup that is easily ionizable to form a positive ion are particularlyuseful in complexing or delivering negatively-charged payloads such aspolynucleotides. Other larger particles or devices may also be preparedfrom the inventive polymers.

In yet another aspect, the invention provides a system for synthesizingand screening a collection of the inventive end-modified polymers. Incertain embodiments, the system takes advantage of techniques known inthe art of automated liquid handling and robotics. The system ofsynthesizing and screening is used with various end-modifiedpoly(beta-amino esters) and end-modified poly(beta-amino amides).Various modifications may be made to these polymers found in thecollection including the bisacrylates, bisacrylamides, or amines used inpreparing the core polymer and the nucleophile used to modify theterminal acrylate or amino moieties. Hundreds to thousands of theinventive end-modified polymers may be synthesized and screened inparallel using the inventive system. In certain embodiments, thepolymers are screened for properties useful in the field of drugdelivery, ability to complex polynucleotides, ability to form particles,biocompatibility, biodegradability, mechanical properties, etc.

DEFINITIONS

Definitions of specific functional groups and chemical terms aredescribed in more detail below. For purposes of this invention, thechemical elements are identified in accordance with the Periodic Tableof the Elements, CAS version, Handbook of Chemistry and Physics, 75^(th)Ed., inside cover, and specific functional groups are generally definedas described therein. Additionally, general principles of organicchemistry, as well as specific functional moieties and reactivity, aredescribed in Organic Chemistry, Thomas Sorrell, University ScienceBooks, Sausalito, 1999; Smith and March March's Advanced OrganicChemistry, 5 th Edition, John Wiley & Sons, Inc., New York, 2001;Larock, Comprehensive Organic Transformations, VCH Publishers, Inc., NewYork, 1989; Carruthers, Some Modern Methods of Organic Synthesis, 3^(rd)Edition, Cambridge University Press, Cambridge, 1987; the entirecontents of each of which are incorporated herein by reference.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.

Isomeric mixtures containing any of a variety of isomer ratios may beutilized in accordance with the present invention. For example, whereonly two isomers are combined, mixtures containing 50:50, 60:40, 70:30,80:20, 90:10, 95:5, 96:4, 97:3, 98:2, 99:1, or 100:0 isomer ratios areall contemplated by the present invention. Those of ordinary skill inthe art will readily appreciate that analogous ratios are contemplatedfor more complex isomer mixtures.

It will be appreciated that the polymers, as described herein, may besubstituted with any number of substituents or functional moieties. Ingeneral, the term “substituted” whether preceded by the term“optionally” or not, and substituents contained in formulas of thisinvention, refer to the replacement of hydrogen radicals in a givenstructure with the radical of a specified substituent. When more thanone position in any given structure may be substituted with more thanone substituent selected from a specified group, the substituent may beeither the same or different at every position. As used herein, the term“substituted” is contemplated to include all permissible substituents oforganic compounds. In a broad aspect, the permissible substituentsinclude acyclic and cyclic, branched and unbranched, carbocyclic andheterocyclic, aromatic and nonaromatic substituents of organiccompounds. For purposes of this invention, heteroatoms such as nitrogenmay have hydrogen substituents and/or any permissible substituents oforganic compounds described herein which satisfy the valencies of theheteroatoms. Furthermore, this invention is not intended to be limitedin any manner by the permissible substituents of organic compounds.Combinations of substituents and variables envisioned by this inventionare preferably those that result in the formation of stable compoundsuseful in the treatment, for example, of infectious diseases orproliferative disorders. The term “stable”, as used herein, preferablyrefers to compounds which possess stability sufficient to allowmanufacture and which maintain the integrity of the compound for asufficient period of time to be detected and preferably for a sufficientperiod of time to be useful for the purposes detailed herein.

The term acyl as used herein refers to a group having the generalformula —C(═O)R, where R is alkyl, alkenyl, alkynyl, aryl, alkoxy,hydroxy, thiol. alkylthioxy, amino, alkylamino, dialkylamino,carbocylic, heterocyclic, or aromatic heterocyclic. An example of anacyl group is acetyl.

The term aliphatic, as used herein, includes both saturated andunsaturated, straight chain (i.e., unbranched), branched, acyclic,cyclic, or polycyclic aliphatic hydrocarbons, which are optionallysubstituted with one or more functional groups. As will be appreciatedby one of ordinary skill in the art, “aliphatic” is intended herein toinclude, but is not limited to, alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, and cycloalkynyl moieties. Thus, as used herein, the term“alkyl” includes straight, branched and cyclic alkyl groups. Ananalogous convention applies to other generic terms such as “alkenyl”,“alkynyl”, and the like. Furthermore, as used herein, the terms “alkyl”,“alkenyl”, “alkynyl”, and the like encompass both substituted andunsubstituted groups. In certain embodiments, as used herein, “loweralkyl” is used to indicate those alkyl groups (cyclic, acyclic,substituted, unsubstituted, branched or unbranched) having 1-6 carbonatoms.

The term alkyl as used herein refers to saturated, straight- orbranched-chain hydrocarbon radicals derived from a hydrocarbon moietycontaining between one and twenty carbon atoms by removal of a singlehydrogen atom. In some embodiments, the alkyl group employed in theinvention contains 1-10 carbon atoms. In another embodiment, the alkylgroup employed contains 1-8 carbon atoms. In still other embodiments,the alkyl group contains 1-6 carbon atoms. In yet another embodiments,the alkyl group contains 1-4 carbons. Examples of alkyl radicalsinclude, but are not limited to, methyl, ethyl, n-propyl, isopropyl,n-butyl, iso-butyl, sec-butyl, sec-pentyl, iso-pentyl, tert-butyl,n-pentyl, neopentyl, n-hexyl, sec-hexyl, n-heptyl, n-octyl, n-decyl,n-undecyl, dodecyl, and the like, which may bear one or moresubstitutents.

The term alkoxy as used herein refers to a saturated (i.e., alkyl-O—) orunsaturated (i.e., alkenyl-O— and alkynyl-O—) group attached to theparent molecular moiety through an oxygen atom. In certain embodiments,the alkyl group contains 1-20 aliphatic carbon atoms. In certain otherembodiments, the alkyl, alkenyl, and alkynyl groups employed in theinvention contain 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl group contains 1-6 aliphatic carbon atoms. In yetother embodiments, the alkyl group contains 1-4 aliphatic carbon atoms.Examples include, but are not limited to, methoxy, ethoxy, propoxy,isopropoxy, n-butoxy, tert-butoxy, i-butoxy, sec-butoxy, neopentoxy,n-hexoxy, and the like.

The term alkenyl denotes a monovalent group derived from a hydrocarbonmoiety having at least one carbon-carbon double bond by the removal of asingle hydrogen atom. In certain embodiments, the alkenyl group employedin the invention contains 1-20 carbon atoms. In some embodiments, thealkenyl group employed in the invention contains 1-10 carbon atoms. Inanother embodiment, the alkenyl group employed contains 1-8 carbonatoms. In still other embodiments, the alkenyl group contains 1-6 carbonatoms. In yet another embodiments, the alkenyl group contains 1-4carbons. Alkenyl groups include, for example, ethenyl, propenyl,butenyl, 1-methyl-2-buten-1-yl, and the like.

The term alkynyl as used herein refers to a monovalent group derivedform a hydrocarbon having at least one carbon-carbon triple bond by theremoval of a single hydrogen atom. In certain embodiments, the alkynylgroup employed in the invention contains 1-20 carbon atoms. In someembodiments, the alkynyl group employed in the invention contains 1-10carbon atoms. In another embodiment, the alkynyl group employed contains1-8 carbon atoms. In still other embodiments, the alkynyl group contains1-6 carbon atoms. Representative alkynyl groups include, but are notlimited to, ethynyl, 2-propynyl (propargyl), 1-propynyl, and the like.

The term alkylamino, dialkylamino, and trialkylamino as used hereinrefers to one, two, or three, respectively, alkyl groups, as previouslydefined, attached to the parent molecular moiety through a nitrogenatom. The term alkylamino refers to a group having the structure —NHR′wherein R′ is an alkyl group, as previously defined; and the termdialkylamino refers to a group having the structure —NR′R″, wherein R′and R″ are each independently selected from the group consisting ofalkyl groups. The term trialkylamino refers to a group having thestructure —NR′R″R′″, wherein R′, R″, and R′″ are each independentlyselected from the group consisting of alkyl groups. In certainembodiments, the alkyl group contain 1-20 aliphatic carbon atoms. Incertain other embodiments, the alkyl group contains 1-10 aliphaticcarbon atoms. In yet other embodiments, the alkyl group contains 1-8aliphatic carbon atoms. In still other embodiments, the alkyl groupcontain 1-6 aliphatic carbon atoms. In yet other embodiments, the alkylgroup contain 1-4 aliphatic carbon atoms. Additionally, R′, R″, and/orR′″ taken together may optionally be —(CH₂)_(k)— where k is an integerfrom 2 to 6. Examples include, but are not limited to, methylamino,dimethylamino, ethylamino, diethylamino, diethylaminocarbonyl,methylethylamino, iso-propylamino, piperidino, trimethylamino, andpropylamino.

The terms alkylthioether and thioalkoxyl refer to a saturated (i.e.,alkyl-S—) or unsaturated (i.e., alkenyl-S— and alkynyl-S—) groupattached to the parent molecular moiety through a sulfur atom. Incertain embodiments, the alkyl group contains 1-20 aliphatic carbonatoms. In certain other embodiments, the alkyl group contains 1-10aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups contain 1-8 aliphatic carbon atoms. In still otherembodiments, the alkyl, alkenyl, and alkynyl groups contain 1-6aliphatic carbon atoms. In yet other embodiments, the alkyl, alkenyl,and alkynyl groups contain 1-4 aliphatic carbon atoms. Examples ofthioalkoxyl moieties include, but are not limited to, methylthio,ethylthio, propylthio, isopropylthio, n-butylthio, and the like.

Some examples of substituents of the above-described aliphatic (andother) moieties of compounds of the invention include, but are notlimited to aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; F; Cl; Br; I; —OH;—NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x)wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substituents are illustratedby the specific embodiments shown in the Examples that are describedherein.

In general, the terms aryl and heteroaryl, as used herein, refer tostable mono- or polycyclic, heterocyclic, polycyclic, andpolyheterocyclic unsaturated moieties having preferably 3-14 carbonatoms, each of which may be substituted or unsubstituted. Substituentsinclude, but are not limited to, any of the previously mentionedsubstitutents, i.e., the substituents recited for aliphatic moieties, orfor other moieties as disclosed herein, resulting in the formation of astable compound. In certain embodiments of the present invention, arylrefers to a mono- or bicyclic carbocyclic ring system having one or twoaromatic rings including, but not limited to, phenyl, naphthyl,tetrahydronaphthyl, indanyl, indenyl, and the like. In certainembodiments of the present invention, the term heteroaryl, as usedherein, refers to a cyclic aromatic radical having from five to ten ringatoms of which one ring atom is selected from S, O, and N; zero, one, ortwo ring atoms are additional heteroatoms independently selected from S,O, and N; and the remaining ring atoms are carbon, the radical beingjoined to the rest of the molecule via any of the ring atoms, such as,for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl, pyrazolyl,imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl, oxadiazolyl,thiophenyl, furanyl, quinolinyl, isoquinolinyl, and the like.

It will be appreciated that aryl and heteroaryl groups can beunsubstituted or substituted, wherein substitution includes replacementof one, two, three, or more of the hydrogen atoms thereon independentlywith any one or more of the following moieties including, but notlimited to: aliphatic; heteroaliphatic; aryl; heteroaryl; arylalkyl;heteroarylalkyl; alkoxy; aryloxy; heteroalkoxy; heteroaryloxy;alkylthio; arylthio; heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I;—OH; —NO₂; —CN; —CF₃; —CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂;—CH₂SO₂CH₃; —C(O)R_(x); —CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x);—OCO₂R_(x); —OCON(R_(x))₂; —N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x),wherein each occurrence of R_(x) independently includes, but is notlimited to, aliphatic, heteroaliphatic, aryl, heteroaryl, arylalkyl, orheteroarylalkyl, wherein any of the aliphatic, heteroaliphatic,arylalkyl, or heteroarylalkyl substituents described above and hereinmay be substituted or unsubstituted, branched or unbranched, cyclic oracyclic, and wherein any of the aryl or heteroaryl substituentsdescribed above and herein may be substituted or unsubstituted.Additional examples of generally applicable substitutents areillustrated by the specific embodiments shown in the Examples that aredescribed herein.

The term carboxylic acid as used herein refers to a group of formula—CO₂H.

The terms halo and halogen as used herein refer to an atom selected fromfluorine, chlorine, bromine, and iodine.

The term haloalkyl denotes an alkyl group, as defined above, having one,two, or three halogen atoms attached thereto and is exemplified by suchgroups as chloromethyl, bromoethyl, trifluoromethyl, and the like.

The term heteroaliphatic, as used herein, refers to aliphatic moietiesthat contain one or more oxygen, sulfur, nitrogen, phosphorus, orsilicon atoms, e.g., in place of carbon atoms. Heteroaliphatic moietiesmay be branched, unbranched, cyclic or acyclic and include saturated andunsaturated heterocycles such as morpholino, pyrrolidinyl, etc. Incertain embodiments, heteroaliphatic moieties are substituted byindependent replacement of one or more of the hydrogen atoms thereonwith one or more moieties including, but not limited to aliphatic;heteroaliphatic; aryl; heteroaryl; arylalkyl; heteroarylalkyl; alkoxy;aryloxy; heteroalkoxy; heteroaryloxy; alkylthio; arylthio;heteroalkylthio; heteroarylthio; —F; —Cl; —Br; —I; —OH; —NO₂; —CN; —CF₃;—CH₂CF₃; —CHCl₂; —CH₂OH; —CH₂CH₂OH; —CH₂NH₂; —CH₂SO₂CH₃; —C(O)R_(x);—CO₂(R_(x)); —CON(R_(x))₂; —OC(O)R_(x); —OCO₂R_(x); —OCON(R_(x))₂;—N(R_(x))₂; —S(O)₂R_(x); —NR_(x)(CO)R_(x), wherein each occurrence ofR_(x) independently includes, but is not limited to, aliphatic,heteroaliphatic, aryl, heteroaryl, arylalkyl, or heteroarylalkyl,wherein any of the aliphatic, heteroaliphatic, arylalkyl, orheteroarylalkyl substituents described above and herein may besubstituted or unsubstituted, branched or unbranched, cyclic or acyclic,and wherein any of the aryl or heteroaryl substituents described aboveand herein may be substituted or unsubstituted. Additional examples ofgenerally applicable substitutents are illustrated by the specificembodiments shown in the Examples that are described herein.

The term heterocyclic, as used herein, refers to an aromatic ornon-aromatic, partially unsaturated or fully saturated, 3- to10-membered ring system, which includes single rings of 3 to 8 atoms insize and bi- and tri-cyclic ring systems which may include aromaticfive- or six-membered aryl or aromatic heterocyclic groups fused to anon-aromatic ring. These heterocyclic rings include those having fromone to three heteroatoms independently selected from oxygen, sulfur, andnitrogen, in which the nitrogen and sulfur heteroatoms may optionally beoxidized and the nitrogen heteroatom may optionally be quaternized. Incertain embodiments, the term heterocylic refers to a non-aromatic 5-,6-, or 7-membered ring or a polycyclic group wherein at least one ringatom is a heteroatom selected from O, S, and N (wherein the nitrogen andsulfur heteroatoms may be optionally oxidized), including, but notlimited to, a bi- or tri-cyclic group, comprising fused six-memberedrings having between one and three heteroatoms independently selectedfrom the oxygen, sulfur, and nitrogen, wherein (i) each 5-membered ringhas 0 to 2 double bonds, each 6-membered ring has 0 to 2 double bonds,and each 7-membered ring has 0 to 3 double bonds, (ii) the nitrogen andsulfur heteroatoms may be optionally oxidized, (iii) the nitrogenheteroatom may optionally be quaternized, and (iv) any of the aboveheterocyclic rings may be fused to an aryl or heteroaryl ring.

The term aromatic heterocyclic, as used herein, refers to a cyclicaromatic radical having from five to ten ring atoms of which one ringatom is selected from sulfur, oxygen, and nitrogen; zero, one, or tworing atoms are additional heteroatoms independently selected fromsulfur, oxygen, and nitrogen; and the remaining ring atoms are carbon,the radical being joined to the rest of the molecule via any of the ringatoms, such as, for example, pyridyl, pyrazinyl, pyrimidinyl, pyrrolyl,pyrazolyl, imidazolyl, thiazolyl, oxazolyl, isooxazolyl, thiadiazolyl,oxadiazolyl, thiophenyl, furanyl, quinolinyl, isoquinolinyl, and thelike. Aromatic heterocyclic groups can be unsubstituted or substitutedwith substituents selected from the group consisting of branched andunbranched alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, thioalkoxy,amino, alkylamino, dialkylamino, trialkylamino, acylamino, cyano,hydroxy, halo, mercapto, nitro, carboxyaldehyde, carboxy,alkoxycarbonyl, and carboxamide.

Specific heterocyclic and aromatic heterocyclic groups that may beincluded in the compounds of the invention include:3-methyl-4-(3-methylphenyl)piperazine, 3 methylpiperidine,4-(bis-(4-fluorophenyl)methyl)piperazine, 4-(diphenylmethyl)piperazine,4-(ethoxycarbonyl)piperazine, 4-(ethoxycarbonylmethyl)piperazine,4-(phenylmethyl)piperazine, 4-(1-phenylethyl)piperazine,4-(1,1-dimethylethoxycarbonyl)piperazine,4-(2-(bis-(2-propenyl)amino)ethyl)piperazine,4-(2-(diethylamino)ethyl)piperazine, 4-(2-chlorophenyl)piperazine,4-(2-cyanophenyl)piperazine, 4-(2-ethoxyphenyl)piperazine,4-(2-ethylphenyl)piperazine, 4-(2-fluorophenyl)piperazine,4-(2-hydroxyethyl)piperazine, 4-(2-methoxyethyl)piperazine,4-(2-methoxyphenyl)piperazine, 4-(2-methylphenyl)piperazine,4-(2-methylthiophenyl)piperazine, 4-(2-nitrophenyl)piperazine,4-(2-nitrophenyl)piperazine, 4-(2-phenylethyl)piperazine,4-(2-pyridyl)piperazine, 4-(2-pyrimidinyl)piperazine,4-(2,3-dimethylphenyl)piperazine, 4-(2,4-difluorophenyl)piperazine,4-(2,4-dimethoxyphenyl)piperazine, 4-(2,4-dimethylphenyl)piperazine,4-(2,5-dimethylphenyl)piperazine, 4-(2,6-dimethylphenyl)piperazine,4-(3-chlorophenyl)piperazine, 4-(3-methylphenyl)piperazine,4-(3-trifluoromethylphenyl)piperazine, 4-(3,4-dichlorophenyl)piperazine,4-3,4-dimethoxyphenyl)piperazine, 4-(3,4-dimethylphenyl)piperazine,4-(3,4-methylenedioxyphenyl)piperazine,4-(3,4,5-trimethoxyphenyl)piperazine, 4-(3,5-dichlorophenyl)piperazine,4-(3,5-dimethoxyphenyl)piperazine,4-(4-(phenylmethoxy)phenyl)piperazine,4-(4-(3,1-dimethylethyl)phenylmethyl)piperazine,4-(4-chloro-3-trifluoromethylphenyl)piperazine,4-(4-chlorophenyl)-3-methylpiperazine, 4-(4-chlorophenyl)piperazine,4-(4-chlorophenyl)piperazine, 4-(4-chlorophenylmethyl)piperazine,4-(4-fluorophenyl)piperazine, 4-(4-methoxyphenyl)piperazine,4-(4-methylphenyl)piperazine, 4-(4-nitrophenyl)piperazine,4-(4-trifluoromethylphenyl)piperazine, 4-cyclohexylpiperazine,4-ethylpiperazine, 4-hydroxy-4-(4-chlorophenyl)methylpiperidine,4-hydroxy-4-phenylpiperidine, 4-hydroxypyrrolidine, 4-methylpiperazine,4-phenylpiperazine, 4-piperidinylpiperazine,4-(2-furanyl)carbonyl)piperazine,4-((1,3-dioxolan-5-yl)methyl)piperazine,6-fluoro-1,2,3,4-tetrahydro-2-methylquinoline, 1,4-diazacylcloheptane,2,3-dihydroindolyl, 3,3-dimethylpiperidine, 4,4-ethylenedioxypiperidine,1,2,3,4-tetrahydroisoquinoline, 1,2,3,4-tetrahydroquinoline,azacyclooctane, decahydroquinoline, piperazine, piperidine, pyrrolidine,thiomorpholine, and triazole.

The term carbamoyl, as used herein, refers to an amide group of theformula —CONH₂.

The term carbonyldioxyl, as used herein, refers to a carbonate group ofthe formula

—O—CO—OR.

The term hydrocarbon, as used herein, refers to any chemical groupcomprising hydrogen and carbon. The hydrocarbon may be substituted orunsubstituted. The hydrocarbon may be unsaturated, saturated, branched,unbranched, cyclic, polycyclic, or heterocyclic. Illustrativehydrocarbons include, for example, methyl, ethyl, n-propyl, iso-propyl,cyclopropyl, allyl, vinyl, n-butyl, tert-butyl, ethynyl, cyclohexyl,methoxy, diethylamino, and the like. As would be known to one skilled inthis art, all valencies must be satisfied in making any substitutions.

The terms substituted, whether preceded by the term “optionally” or not,and substituent, as used herein, refer to the ability, as appreciated byone skilled in this art, to change one functional group for anotherfunctional group provided that the valency of all atoms is maintained.When more than one position in any given structure may be substitutedwith more than one substituent selected from a specified group, thesubstituent may be either the same or different at every position. Thesubstituents may also be further substituted (e.g., an aryl groupsubstituent may have another substituent off it, such as another arylgroup, which is further substituted with fluorine at one or morepositions).

The term thiohydroxyl or thiol, as used herein, refers to a group of theformula —SH.

The following are more general terms used throughout the presentapplication:

“Animal”: The term animal, as used herein, refers to humans as well asnon-human animals, including, for example, mammals, birds, reptiles,amphibians, and fish. Preferably, the non-human animal is a mammal(e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, aprimate, or a pig). An animal may be a domesticated animal. An animalmay be a transgenic animal.

“Associated with”: When two entities are “associated with” one anotheras described herein, they are linked by a direct or indirect covalent ornon-covalent interaction. In certain embodiments, the association iscovalent. In other embodiments, the association is non-covalent.Desirable non-covalent interactions include hydrogen bonding, van derWaals interactions, hydrophobic interactions, magnetic interactions,electrostatic interactions, etc.

“Biocompatible”: The term “biocompatible”, as used herein is intended todescribe compounds that are not toxic to cells. Compounds are“biocompatible” if their addition to cells in vitro results in less thanor equal to 20% cell death, and their administration in vivo does notinduce inflammation or other such adverse effects.

“Biodegradable”: As used herein, “biodegradable” compounds are thosethat, when introduced into cells, are broken down by the cellularmachinery or by hydrolysis into components that the cells can eitherreuse or dispose of without significant toxic effects on the cells(i.e., fewer than about 20% of the cells are killed when the componentsare added to cells in vitro). The components preferably do not induceinflammation or other adverse effects in vivo. In certain preferredembodiments, the chemical reactions relied upon to break down thebiodegradable compounds are uncatalyzed. For example, the inventivematerials may be broken down in part by the hydrolysis of the esterbonds found in cross-linked material.

“Peptide” or “protein”: According to the present invention, a “peptide”or “protein” comprises a string of at least three amino acids linkedtogether by peptide bonds. The terms “protein” and “peptide” may be usedinterchangeably. Peptide may refer to an individual peptide or acollection of peptides. Inventive peptides preferably contain onlynatural amino acids, although non-natural amino acids (i.e., compoundsthat do not occur in nature but that can be incorporated into apolypeptide chain) and/or amino acid analogs as are known in the art mayalternatively be employed. Also, one or more of the amino acids in aninventive peptide may be modified, for example, by the addition of achemical entity such as a carbohydrate group, a phosphate group, afarnesyl group, an isofarnesyl group, a fatty acid group, a linker forconjugation, functionalization, or other modification, etc. In apreferred embodiment, the modifications of the peptide lead to a morestable peptide (e.g., greater half-life in vivo). These modificationsmay include cyclization of the peptide, the incorporation of D-aminoacids, etc. None of the modifications should substantially interferewith the desired biological activity of the peptide.

“Polynucleotide” or “oligonucleotide”: Polynucleotide or oligonucleotiderefers to a polymer of nucleotides. Typically, a polynucleotidecomprises at least three nucleotides. The polymer may include naturalnucleosides (i.e., adenosine, thymidine, guanosine, cytidine, uridine,deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine),nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine,pyrrolo-pyrimidine, 3-methyl adenosine, C5-propynylcytidine,C5-propynyluridine, C5-bromouridine, C5-fluorouridine, C5-iodouridine,C5-methylcytidine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine,8-oxoguanosine, O(6)-methylguanine, and 2-thiocytidine), chemicallymodified bases, biologically modified bases (e.g., methylated bases),intercalated bases, modified sugars (e.g., 2′-fluororibose, ribose,2′-deoxyribose, arabinose, and hexose), or modified phosphate groups(e.g., phosphorothioates and 5′-N-phosphoramidite linkages).

“Small molecule”: As used herein, the term “small molecule” refers toorganic compounds, whether naturally-occurring or artificially created(e.g., via chemical synthesis) that have relatively low molecular weightand that are not proteins, polypeptides, or nucleic acids. Typically,small molecules have a molecular weight of less than about 1500 g/mol.Also, small molecules typically have multiple carbon-carbon bonds. Knownnaturally-occurring small molecules include, but are not limited to,penicillin, erythromycin, taxol, cyclosporin, and rapamycin. Knownsynthetic small molecules include, but are not limited to, ampicillin,methicillin, sulfamethoxazole, and sulfonamides.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1. (A) Synthesis of acrylate-terminated C32 polymer (C32-Ac). (B)Synthesis of end-modified C32 polymers (C32-X). (C) Amine cappingmolecules. (D) Structures of the acrylate-terminated C20 and D60poly(beta-amino esters) used to demonstrate the combined effects ofinterior polymer sequence and end-modification on DNA delivery.End-modified derivatives of AA28 were synthesized and tested for siRNAdelivery.

FIG. 2. Transfection of COS-7 cells. The relative light units (RLU) ofluciferase reporter protein expressed are shown for each end-modifiedC32 polymer (C32-X) at five polymer:DNA weight ratios (10:1, 20:1, 30:1,60:1, and 100:1).

FIG. 3. Cytotoxicity levels of end-modified C32 polymers (C32-X)measured using the MTT assay. PE1, C32+, C32-Ac, and free DNA toxicityare shown the far right.

FIG. 4. Polymer-DNA binding measured using a PicoGreen assay.Fluorescence reductions relative to free DNA (RF) are shown for eachend-modified C32-polymer (C32-X) at the optimal transfecting polymer:DNAratio for each polymer.

FIG. 5. Polymer-DNA complex size measured by dynamic light scattering.The effective diameter of complexes are shown for each end-modified C32polymer (C32-X) at the optimal transfecting polymer:DNA ratio for eachpolymer. In general, diamine-capped polymers form smaller polymer-DNAcomplexes than C32+. The smallest particle size in serum is 86 nm. ForC32+, it is 152 nm.

FIG. 6. Plasmid DNA uptake into COS-7 cells. DNA uptake levels are shownin number of plasmids per COS-7 DNA fluorescence. Diamine-cappedpolymers increase DNA uptake five times over C32+ and PEI.

FIG. 7. Transfection of COS-7 (blue bars) and HepG2 (red bars) byend-modified C32, D60, and C20 polymers at a 20:1 polymer:DNA ratio.

FIG. 8. siRNA delivery with AA28 poly(beta-amino esters). Percentknockdown of firefly luciferase in HeLa cells is shown for eachend-modified AA28 polymer (AA28-X) at its optimal polymer:siRNA ratio.Certain end-modified polymers have been found to be as effective asLipo2000. Higher positive charge density at the ends results in highersilencing.

FIG. 9. Transfection of cells by end-modified C32 at 100:1 polymer:DNAand 20:1 polymer:DNA ratios.

FIG. 10. Polymer-DNA complex size and plasmid DNA uptake forend-modified C32 (C32-36; C32-52; C32-106).

DETAILED DESCRIPTION OF CERTAIN PREFERRED EMBODIMENTS OF THE INVENTION

The present invention provides novel end-modified poly(beta-aminoesters), poly(beta-amino amides), and other polymers. In certainembodiments, the inventive polymers are prepared by reacting anacrylate-terminated poly(beta-amino ester) with a nucleophile undersuitable conditions to have the nucleophile added to the terminalacrylate units of the polymer. In certain embodiments, the inventivepolymers are prepared by reacting an acrylate-terminated poly(beta-aminoamides) with a nucleophile under suitable conditions to have thenucleophile added to the terminal acrylate units of the polymer. Incertain embodiments, the inventive polymers are prepared by reacting anamine-terminated poly(beta-amino ester) with a electrophile undersuitable conditions to have the electrophile added to the terminal aminounits of the polymer. In certain embodiments, the inventive polymers areprepared by reacting an amine-terminated poly(beta-amino amides) with anelectrophile under suitable conditions to have the electrophile react tothe terminal amino units of the polymer. The end-modified polymers areuseful in many different areas including drug delivery and thebiomedical arts. The invention also provides methods of preparing theinventive end-modified polymers, screening these polymers for specificproperties, and using these materials in the medical field andnon-medical fields. In certain embodiments, a system is provided forpreparing and screening a library of the inventive end-modified polymersin parallel. High-throughput techniques and devices may be used in thissystem. The invention also provides compositions including the inventiveend-modified polymers (e.g., drug delivery devices (e.g., complexes,nanoparticles, microparticles, macroparticles, capsules, tablets),microdevices, nanodevices, tissue engineering scaffolds, plastics,films, biomedical devices, etc.)

In certain embodiments, the inventive end-modified poly(beta-aminoesters) are prepared from poly(beta-amino esters). The poly(beta-aminoester) is modified at its termini with a nucleophilic reagent.Preferably, the poly(beta-amino ester) is terminated with anelectrophilic moiety such as an acrylate or methacrylate. Suchα,β-unsaturated esters are susceptible to 1,4-addition by a nucleophilicreagent thereby resulting in the end-modified poly(beta-amino ester). Incertain embodiments, the reaction conditions and reagent are such thatthe 1,4-addition to the α,β-unsaturated carbonyl is favored over the1,2-addition. Poly(beta-amino esters) and the preparation of thesepolymers are described in U.S. patent applications U.S. Ser. No.11/099,886, filed Apr. 6, 2005; U.S. Ser. No. 10/446,444, filed May 28,2003; U.S. Ser. No. 09/969,431, filed Oct. 2, 2001; U.S. Ser. No.60/305,337, filed Jul. 13, 2001; and U.S. Ser. No. 60/239,330, filedOct. 10, 2000; each of which is incorporated herein by reference. Thesepolymers are prepared by the conjugate addition of a primary amine or abis(secondary amine) to diacrylates. Preferably, the polymers areterminated with an acrylate unit; therefore, the preparation of thepolymer is done with an excess of acrylate. These polymers have alreadybeen shown to be particularly useful in drug delivery such as thedelivery of polynucleotides due to the presence of tertiary amines inthe backbone of the polymer. These polymers and their end-modifiedvariants are also useful in the medical and non-medical arts because ofthe biodegradable nature of the ester linkages in the polymers. Theinventive end-modified poly(beta-amino esters) are prepared by theaddition of a nucleophile to the end(s) of the polymer. The resultingend-modified polymers are useful in a variety of applications includingthe medical and non-medical fields.

In other embodiments, poly(beta-amino amides) are similarly end-modifiedas described above for poly(beta-amino esters). In yet otherembodiments, other polymers with reactive terminal moieties areend-modified by the inventive system. The resulting end-modifiedpolymers, method of preparing the end-modified polymers, methods ofusing the end-modified polymers, and compositions comprising theend-modified polymers are considered part of the present invention.

Poly(beta-amino esters)

Poly(beta-amino esters) are used as the starting material in preparingthe inventive end-modified poly(beta-amino esters). Any size of polymerof poly(beta-amino esters) may be useful in the preparation of theinventive crosslinked materials. In certain embodiments, the molecularweights of the polymers range from 1,000 g/mol to over 100,000 g/mol,more preferably from 1,000 g/mol to 50,000 g/mol. In certainembodiments, the molecular weights of the polymers range from 500 g/molto 10,000 g/mol. In other embodiments, the molecular weights of thepolymers range from 1,000 g/mol to 25,000 g/mol. In certain embodiments,the molecular weights of the polymers range from 2,000 g/mol to 15,000g/mol. In certain embodiments, the average molecular weight of thepolymer is approximately 1,000 g/mol, 2,000 g/mol, 3,000 g/mol, 4,000g/mol, 5,000 g/mol, 6,000 g/mol, 7,000 g/mol, 8,000 g/mol, 9,000 g/mol,10,000 g/mol, 11,000 g/mol, 12,000 g/mol, 13,000 g/mol, 14,000 g/mol,15,000 g/mol, 16,000 g/mol, 17,000 g/mol, 18,000 g/mol, 19,000 g/mol, or20,000 g/mol. In certain embodiments, even smaller polymers are used. Inother embodiments, even larger polymers are used. In a particularlypreferred embodiment, the polymers are relatively non-cytotoxic. Inanother particularly preferred embodiment, the polymers arebiocompatible and biodegradable. In another embodiment, the polymers ofthe present invention have pK_(a)s in the range of 5.5 to 7.5, morepreferably between 6.0 and 7.0. In another embodiment, the polymer maybe designed to have a desired pK_(a) between 3.0 and 9.0, morepreferably between 5.0 and 8.0. In certain embodiments, the polymer hasmore than one acidic and/or basic moiety resulting in more than one pKa.

The poly(beta-amino esters) useful in preparing the inventiveend-modified polymers include a terminal electrophilic group suitablefor addition of the nucleophile. The polymers typically have an acrylateor methacrylate group at each end of the polymer. Acrylate-terminatedpolymers are easily prepared by using an excess of acrylate in thesynthesis of the poly(beta-amino ester). In certain embodiments, thepolymer ends with a functional group of formula:

In other embodiments, the polymer ends with a functional group offormula:

The acrylate-terminated poly(beta-amino ester) is reacted with anucleophile to yield an end-modified polymer of formulae:

wherein

A and B are linkers which may be any substituted or unsubstituted,branched or unbranched, cyclic or acyclic aliphatic or heteroaliphaticmoiety; or substituted or unsubstituted aryl or heteroaryl moieties;

each of R₁, R₂, R₃, and R₄ are independently a hydrogen; halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and

each X is independently O, S, NH, or NR_(X), wherein R_(X) is halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and salts thereof.

The poly(beta-amino esters) useful in preparing the inventiveend-modified polymers include a terminal nucleophile group suitable forreacting with an electrophile. In certain embodiments, the polymers havean amino group at each end of the polymer. Amine-terminated polymers areeasily prepared by using an excess of amine in the synthesis of thepoly(beta-amino ester). In certain embodiments, the polymer ends with afunctional group of formula —NH₂. In other embodiments, the polymer endswith a functional group of formula —NR′H, wherein R′ is substituted orunsubstituted, branched or unbranched, cyclic or acyclic aliphatic orheteroaliphatic; substituted or unsubstituted acyl; or substituted orunsubstituted aryl or heteroaryl. In certain embodiments, R′ is C₁-C₆alkyl.

The amine-terminated poly(beta-amino ester) is reacted with anelectrophile to yield an end-modified polymer of formulae:

wherein

A and B are linkers which may be any substituted or unsubstituted,branched or unbranched, cyclic or acyclic aliphatic or heteroaliphaticmoiety; or substituted or unsubstituted aryl or heteroaryl moieties;

each of R₁, R₂, R₃, and R₄ are independently a hydrogen; halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and

each X is independently O, S, NH, or NR_(X), wherein R_(X) is halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and salts thereof

In certain embodiments, an acrylate-terminated poly(beta-amino amide) isreacted with a nucleophile to yield an end-modified polymer of formulae:

wherein

A and B are linkers which may be any substituted or unsubstituted,branched or unbranched, cyclic or acyclic aliphatic or heteroaliphaticmoiety; or substituted or unsubstituted aryl or heteroaryl moieties;

each R′ is independently a hydrogen; branched or unbranched, substitutedor unsubstituted, cyclic or acyclic aliphatic; branched or unbranched,substituted or unsubstituted, cyclic or acyclic heteroaliphatic;branched or unbranched, substituted or unsubstituted, cyclic or acyclicacyl; substituted or unsubstituted aryl; or substituted or unsubstitutedheteroaryl;

each of R₁, R₂, R₃, and R₄ are independently a hydrogen; halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and

each X is independently O, S, NH, or NR_(X), wherein R_(X) is halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and salts thereof.

In other embodiments, an amine-terminated poly(beta-amino amide) isreacted with an electrophile to yield an end-modified polymer offormulae:

wherein

A and B are linkers which may be any substituted or unsubstituted,branched or unbranched, cyclic or acyclic aliphatic or heteroaliphaticmoiety; or substituted or unsubstituted aryl or heteroaryl moieties;

each R′ is independently a hydrogen; branched or unbranched, substitutedor unsubstituted, cyclic or acyclic aliphatic; branched or unbranched,substituted or unsubstituted, cyclic or acyclic heteroaliphatic;branched or unbranched, substituted or unsubstituted, cyclic or acyclicacyl; substituted or unsubstituted aryl; or substituted or unsubstitutedheteroaryl;

each of R₁, R₂, R₃, and R₄ are independently a hydrogen; halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and

each X is independently O, S, NH, or NR_(X), wherein R_(X) is halogen;branched or unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and salts thereof.

In certain embodiments, all R′ are hydrogen. In other embodiments, allR′ are C₁-C₆ alkyl. In yet other embodiments, all R′ are methyl. Incertain embodiments, all R′ are acyl.

The linkers A and B are each a chain of atoms covalently linking theamino groups and ester groups, respectively. These linkers may containcarbon atoms or heteroatoms (e.g., nitrogen, oxygen, sulfur, etc.).Typically, these linkers are 1 to 30 atoms long, more preferably 1-15atoms long. The linkers may contain cyclic structures including aromaticand non-aromatic structures. The linker may include aromatic structuresincluding aryl and heteroaryl groups. The linkers may be substitutedwith various substituents including, but not limited to, hydrogen,alkyl, alkenyl, alkynyl, amino, alkylamino, dialkylamino, trialkylamino,hydroxyl, alkoxy, halogen, aryl, heterocyclic, aromatic heterocyclic,cyano, amide, carbamoyl, carboxylic acid, ester, thioether,alkylthioether, thiol, and ureido groups. In certain embodiments, thelinker A or B is unbranched alkylidene moieties of 1-20 carbons. Incertain embodiments, the linker A or B is unbranched alkylidene moietiesof 1-12 carbons. In certain embodiments, the linker A or B is unbranchedalkylidene moieties of 1-6 carbons. In certain embodiments, the linker Aor B is branched alkylidene moieties of 1-20 carbons. In certainembodiments, the linker A or B is branched alkylidene moieties of 1-12carbons. In certain embodiments, the linker A or B is branchedalkylidene moieties of 1-6 carbons. In certain embodiments, the linker Aor B is a polyethylene glycol linker. In certain embodiments, the linkerA or B is a polyethylene glycol linker of 3-25 atoms in length. Incertain embodiments, the linker A or B is a polyethylene glycol linkerof 3-18 atoms in length. As would be appreciated by one of skill in thisart, each of these groups may in turn be substituted.

The groups R₁, R₂, R₃, and R₄ may be any chemical groups including, butnot limited to, hydrogen atoms, alkyl, alkenyl, alkynyl, amino,alkylamino, dialkylamino, trialkylamino, hydroxyl, alkoxy, halogen,aryl, heterocyclic, aromatic heterocyclic, cyano, amide, carbamoyl,carboxylic acid, ester, alkylthioether, thiol, and ureido groups. Incertain embodiments, R₁ and R₂ are the same. In certain embodiments, R₃and R₄ are the same. In other embodiments, R₃ and R₄ are different. Incertain embodiments, R₁, R₃, and R₄ are the same. In other embodiments,R₃ and R₄ are aliphatic. In certain other embodiments, R₃ and R₄ areheteroaliphatic. In yet other embodiments, R₃ and R₄ are aryl. In stillother embodiments, R₃ and R₄ are heteroaryl. In certain embodiments, R₃and R₄ are independently C₁-C₁₂ alkyl. In other embodiments, R₃ and R₄are C₁-C₆ alkyl. In certain embodiments, R₃ and R₄ are independentlyC₂-C₁₂ alkenyl. In other embodiments, R₃ and R₄ are C₂-C₆ alkenyl. Incertain embodiments, R₃ and R₄ are independently C₂-C₁₂ alkynyl. Inother embodiments, R₃ and R₄ are C₁-C₆ alkynyl.

In certain embodiments, the amine used to prepare the poly(beta-aminoester) is a cyclic secondary diamine. End-modified version of such apoly(beta-amino ester) are of the formula:

R₁ and R₂ form a cyclic structure along with the two N atoms and thelinker A.

In other embodiments, the groups R₁ and/or R₂ are covalently bonded tolinker A to form one or two cyclic structures. The end-modified polymersof the present embodiment are generally represented by the formula belowin which both R₁ and R₂ are bonded to linker A to form two cyclicstructures:

In certain embodiments, the amine used to prepare the poly(beta-aminoamide) is a cyclic secondary diamine. End-modified version of such apoly(beta-amino amide) are of the formula:

R₁ and R₂ form a cyclic structure along with the two N atoms and thelinker A.

In other embodiments, the groups R₁ and/or R₂ are covalently bonded tolinker A to form one or two cyclic structures. The end-modified polymersof the present embodiment are generally represented by the formula belowin which both R₁ and R₂ are bonded to linker A to form two cyclicstructures:

In certain embodiments, X is O. In certain embodiments, X is S. In otherembodiments, X is NH. In yet other embodiments, X is NR_(X). In certainembodiments, X is NR_(X), wherein R_(X) is C₁-C₆ alkyl. In certainparticular embodiments, NR_(X) is NMe. In other embodiments, NR_(X) isNEt. In certain embodiments, both X are the same. In certainembodiments, both X are NRX (e.g., NH). In other embodiments, both X areO. In other embodiments, the X are different. For example, in certainembodiments, one X is O, and the other is NR_(X) (e.g., NH). In certainembodiments, one X is S, and the other is O.

In certain embodiments, R₃ and R₄ are wherein m is an integer between 1and 20, inclusive. In certain embodiments, m is an integer between 2 and15, inclusive. In yet other embodiments, m is an integer between 2 and12, inclusive. In other embodiments, m is an integer between 2 and 10,inclusive. In other embodiments, m is an integer between 2 and 6,inclusive. In still other embodiments, m is an integer between 2 and 3,inclusive.

In certain embodiments, R₃ and R₄ are

wherein m is an integer between 1 and 20, inclusive. In certainembodiments, m is an integer between 2 and 15, inclusive. In yet otherembodiments, m is an integer between 2 and 12, inclusive. In otherembodiments, m is an integer between 2 and 10, inclusive. In otherembodiments, m is an integer between 2 and 6, inclusive. In still otherembodiments, m is an integer between 2 and 3, inclusive.

In certain embodiments, R₃ and R₄ are

wherein m is an integer between 1 and 20, inclusive. In certainembodiments, m is an integer between 2 and 15, inclusive. In yet otherembodiments, m is an integer between 2 and 12, inclusive. In otherembodiments, m is an integer between 2 and 10, inclusive. In otherembodiments, m is an integer between 2 and 6, inclusive. In still otherembodiments, m is an integer between 1 and 3, inclusive.

In certain embodiments, R₃ and R₄ are wherein m is an integer between 0and 20, inclusive. In certain embodiments, m is an integer between 0 and15, inclusive. In yet other embodiments, m is an integer between 0 and12, inclusive. In other embodiments, m is an integer between 0 and 10,inclusive. In other embodiments, m is an integer between 0 and 6,inclusive. In still other embodiments, m is an integer between 0 and 3,inclusive. In certain embodiments, n is 0, 1, 2, 3, 4, 5, or 6.

In certain embodiments, R₃ and R₄ are wherein m is an integer between 0and 20, inclusive. In certain embodiments, m is an integer between 1 and15, inclusive. In yet other embodiments, m is an integer between 1 and12, inclusive. In other embodiments, m is an integer between 1 and 10,inclusive. In other embodiments, m is an integer between 0 and 6,inclusive. In still other embodiments, m is an integer between 0 and 3,inclusive. In certain embodiments, m is 0, 1, 2, 3, 4, 5, or 6. Incertain embodiments, the terminal amino group of R₃ and/or R₄ isprotected, alkylated (e.g., C₁-C₁₂ alkyl), acylated (e.g., acetyl), orotherwise modified.

In certain embodiments, R₃ and R₄ are

wherein m is an integer between 0 and 20, inclusive. In certainembodiments, m is an integer between 1 and 15, inclusive. In yet otherembodiments, m is an integer between 1 and 12, inclusive. In otherembodiments, m is an integer between 1 and 10, inclusive. In otherembodiments, m is an integer between 0 and 6, inclusive. In still otherembodiments, m is an integer between 0 and 3, inclusive. In certainembodiments, m is 0, 1, 2, 3, 4, 5, or 6. In certain embodiments, theterminal hydroxyl group of R₃ and/or R₄ is protected, alkylated (e.g.,C₁-C₁₂ alkyl), acylated (e.g., acetyl), or otherwise modified.

In certain embodiments, R₃ and R₄ are wherein m is an integer between 0and 20, inclusive. In certain embodiments, m is an integer between 1 and15, inclusive. In yet other embodiments, m is an integer between 1 and12, inclusive. In other embodiments, m is an integer between 1 and 10,inclusive. In other embodiments, m is an integer between 0 and 6,inclusive. In still other embodiments, m is an integer between 0 and 3,inclusive. In certain embodiments, m is 0, 1, 2, 3, 4, 5, or 6.

In certain embodiments, R₃ and R₄ are

wherein n, m, and p are each independently an integer between 0 and 20,inclusive; and V is —O—, —S—, —NH—, —NR_(V)—, or C(R_(V))₂, whereinR_(V) is hydrogen, hydroxyl, C₁₋₆aliphatic, C₁₋₆heteroaliphatic,C₁₋₆alkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, aryl, heteroaryl,thiol, alkylthioxy, or acyl. In certain embodiments, n, m, and p areeach independently an integer between 1 and 15, inclusive. In yet otherembodiments, n, m, and p are each independently an integer between 1 and12, inclusive. In other embodiments, n, m, and p are each independentlyan integer between 1 and 10, inclusive. In other embodiments, n, m, andp are each independently an integer between 0 and 6, inclusive. In stillother embodiments, n, m, and p are each independently an integer between0 and 3, inclusive. In certain embodiments, n, m, and p are eachindependently 0, 1, 2, 3, 4, 5, or 6.

In certain embodiments, R₃ and R₄ are

wherein n, m, and p are each independently an integer between 0 and 20,inclusive; and V is —O—, —S—, —NH—, —NR_(V)—, or C(R_(V))₂, whereinR_(V) is hydrogen, hydroxyl, C₁₋₆aliphatic, C₁₋₆heteroaliphatic,C₁₋₆alkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, aryl, heteroaryl,thiol, alkylthioxy, or acyl. In certain embodiments, n, m, and p areeach independently an integer between 1 and 15, inclusive. In yet otherembodiments, n, m, and p are each independently an integer between 1 and12, inclusive. In other embodiments, n, m, and p are each independentlyan integer between 1 and 10, inclusive. In other embodiments, n, m, andp are each independently an integer between 0 and 6, inclusive. In stillother embodiments, n, m, and p are each independently an integer between0 and 3, inclusive. In certain embodiments, n, m, and p are eachindependently 0, 1, 2, 3, 4, 5, or 6. In certain embodiments, theterminal hydroxyl group of R₃ and/or R₄ is protected, alkylated (e.g.,C₁-C₁₂ alkyl), acylated (e.g., acetyl), or otherwise modified.

In certain embodiments, R₃ and R₄ are wherein n, m, and p are eachindependently an integer between 0 and 20, inclusive; and V is —O—, —S—,—NH—, —NR_(V)—, or C(R_(V))₂, wherein R_(V) is hydrogen, hydroxyl,C₁₋₆aliphatic, C₁₋₆heteroaliphatic, C₁₋₆alkoxy, amino, C₁₋₆alkylamino,di(C₁₋₆alkyl)amino, aryl, heteroaryl, thiol, alkylthioxy, or acyl. Incertain embodiments, n, m, and p are each independently an integerbetween 1 and 15, inclusive. In yet other embodiments, n, m, and p areeach independently an integer between 1 and 12, inclusive. In otherembodiments, n, m, and p are each independently an integer between 1 and10, inclusive. In other embodiments, n, m, and p are each independentlyan integer between 0 and 6, inclusive. In still other embodiments, n, m,and p are each independently an integer between 0 and 3, inclusive. Incertain embodiments, n, m, and p are each independently 0, 1, 2, 3, 4,5, or 6. In certain embodiments, the terminal amino group of R₃ and/orR₄ is protected, alkylated (e.g., C₁-C₁₂ alkyl), acylated (e.g.,acetyl), or otherwise modified.

In certain embodiments, R₃ and R₄ are

wherein n, m, and p are each independently an integer between 0 and 20,inclusive; and V is —O—, —S—, —NH—, —NR_(V)—, or C(R_(V))₂, whereinR_(V) is hydrogen, hydroxyl, C₁₋₆aliphatic, C₁₋₆heteroaliphatic,C₁₋₆alkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, aryl, heteroaryl,thiol, alkylthioxy, or acyl. In certain embodiments, n, m, and p areeach independently an integer between 1 and 15, inclusive. In yet otherembodiments, n, m, and p are each independently an integer between 1 and12, inclusive. In other embodiments, n, m, and p are each independentlyan integer between 1 and 10, inclusive. In other embodiments, n, m, andp are each independently an integer between 0 and 6, inclusive. In stillother embodiments, n, m, and p are each independently an integer between0 and 3, inclusive. In certain embodiments, n, m, and p are eachindependently 0, 1, 2, 3, 4, 5, or 6.

In certain embodiments, R₃ and R₄ are selected from the group consistingof:

In certain other embodiments, R₃ and R₄ are selected from the groupconsisting of:

In certain other embodiments, R₃ and R₄ are selected from the groupconsisting of:

In certain embodiments, X is O. In other embodiments, X is NH. In yetother embodiments, X is NR_(X). In certain embodiments, X is NR_(X),wherein R_(X) is C₁-C₆ alkyl. In certain particular embodiments, NR_(X)is NMe. In other embodiments, NR_(X) is NEt.

In the inventive polymers, n is an integer ranging from 5 to 10,000,more preferably from 10 to 500.

In certain embodiments, the end-modified polymers are of the formulaebelow wherein bismethyacrylate units have been used to prepare themethacrylate-terminated poly(beta-amino ester) which are subsequentlyend-modified with a nucleophile:

wherein R₁, R₂, R₃, R₄, X, A, and B are defined in the genera, classes,subclasses, and species as above.

In another embodiment, the diacrylate unit in the poly(beta-amino ester)is chosen from the following group of diacrylate units (A-PP):

In another embodiment, the diacrylate unit of the polymer is chosen fromthe following group of diacrylate units (A′-G′):

Particularly preferred diacrylate units include A, B′, C, C′, J, U, AA,PP, and L.

In another embodiment, the amine used in the preparation of thepoly(beta-amino ester) is chosen from the following group of amines(1′-20′):

As would be appreciated by one of skill in this art, these amines mayalso be used to end-modify acrylate-terminated poly(beta-amino esters).

In another embodiment, the amine used in the preparation of thepoly(beta-amino ester) is chosen from the following group of amines(1-94):

In certain embodiments, the polymers include amine unit 1, 8, 25, 28,31, 32, 40, 58, 60, 73, 87, 91, and 12. As would be appreciated by oneof skill in this art, these amines may also be used to end-modifyacrylate-terminated poly(beta-amino esters).

In certain embodiments, the core poly(beta-amino ester) beforemodification is B′14′, G′5′, A′14′, C′5′, G′7′, G′10′, G′12′, C36, M17,KK89, and D94. In certain embodiments, the core poly(beta-amino ester)before end-modification is C20, C32, D60, or AA28. In certainembodiments, the end-modified poly(beta-amino ester) is C32 terminatedwith amine 36, 52, 95, or 110.

In certain embodiments, R₁ is a branched or unbranched, substituted orunsubstituted aliphatic moiety. In certain embodiments, R₁ is analiphatic moiety substituted with hydroxyl or alkoxy moieties. Incertain embodiments, R₁ is an alkyl moiety substituted with hydroxyl oralkoxy moieties. In certain embodiments, R₁ is an alkyl moietysubstituted with an amino, alkylamino, or dialkylamino moiety. Incertain embodiments, R₁ is an alkyl moiety substituted with a guanidine,ortho-ester, phosphate, or phospho-lipid moiety. In certain embodiments,R₁ is an alkyl moiety substituted with a halogen. In certainembodiments, R₁ is an alkyl moiety substituted with a heterocylic moiety(e.g., triazines, piperidines, piperazines, aziridines, etc.). In otherembodiments, R₁ is an alkyl moiety substituted with a heteroaryl moiety(e.g., pyrindinyl, triazines, furanyl, imidazolyl). In otherembodiments, R₁ is a branched or unbranched, substituted orunsubstituted heteroaliphatic moiety. In certain embodiments, R₁ is asubstituted or unsubstituted aryl moiety (e.g., phenyl, naphthyl, etc.).In other embodiments, R₁ is a substituted or unsubstituted heteroarylmoiety (e.g., imidazoyl, thiazolyl, oxazolyl, pyridinyl, etc.). Incertain embodiments, R₁ is C₁-C₂₀ alkyl. In other embodiments, R₁ isC₁-C₁₂ alkyl. In other embodiments, R₁ is C₁-C₆ alkyl. In certainembodiments, R₁ is methyl. In other embodiments, R₁ is ethyl.

In certain embodiments, R₁ is selected from the group consisting of:

In certain embodiments, R₂ is a branched or unbranched, substituted orunsubstituted aliphatic moiety. In certain embodiments, R₂ is analiphatic moiety substituted with hydroxyl or alkoxy moieties. Incertain embodiments, R₂ is an alkyl moiety substituted with hydroxyl oralkoxy moieties. In certain embodiments, R₂ is an alkyl moietysubstituted with an amino, alkylamino, or dialkylamino moiety. Incertain embodiments, R₂ is an alkyl moiety substituted with a guanidine,ortho-ester, phosphate, or phospho-lipid moiety. In certain embodiments,R₂ is an alkyl moiety substituted with a halogen. In certainembodiments, R₂ is an alkyl moiety substituted with a heterocylic moiety(e.g., triazines, piperidines, piperazines, aziridines, etc.). In otherembodiments, R₂ is an alkyl moiety substituted with a heteroaryl moiety(e.g., pyrindinyl, triazines, furanyl, imidazolyl). In otherembodiments, R₂ is a branched or unbranched, substituted orunsubstituted heteroaliphatic moiety. In certain embodiments, R₂ is asubstituted or unsubstituted aryl moiety (e.g., phenyl, naphthyl, etc.).In other embodiments, R₂ is a substituted or unsubstituted heteroarylmoiety (e.g., pyridinyl, triazines, imidazoyl, thiazolyl, oxazolyl,pyridinyl, etc.). In certain embodiments, R₂ is C₁-C₂₀ alkyl. In otherembodiments, R₂ is C₁-C₁₂ alkyl. In other embodiments, R₂ is C₁-C₆alkyl. In certain embodiments, R₂ is methyl. In other embodiments, R₂ isethyl.

In certain embodiments, one or both of the linkers A and B are linkerscontaining only carbon, oxygen, and hydrogen atoms. In certainembodiments, one or both of the linkers A and B are linkers containingonly carbon and hydrogen atoms. In certain embodiments, one or both ofthe linkers A and B are linkers containing only carbon and halogenatoms. In one embodiment, one or both of the linkers A and B arepolyethylene linkers. In another particularly preferred embodiment, oneor both of the linkers A and B are polyethylene glycol linkers. Otherbiocompatible, biodegradable linkers may be used as one or both of thelinkers A and B.

In certain embodiments, A is

wherein n is an integer between 1 and 20, inclusive. In certainembodiments, n is an integer between 1 and 15, inclusive. In otherembodiments, n is an integer between 1 and 10, inclusive. In yet otherembodiments, n is an integer between 1 and 6, inclusive. In certainembodiments, n is 2. In other embodiments, n is 3. In yet otherembodiments, n is 4. In still other embodiments, n is 6.

In certain embodiments, A is

wherein n is an integer between 1 and 20, inclusive. In certainembodiments, n is an integer between 1 and 15, inclusive. In otherembodiments, n is an integer between 1 and 10, inclusive. In yet otherembodiments, n is an integer between 1 and 6, inclusive. In certainembodiments, n is 1. In certain embodiments, n is 2. In otherembodiments, n is 3. In yet other embodiments, n is 4. In still otherembodiments, n is 6.

In certain embodiments, A is

wherein n is an integer between 1 and 20, inclusive; and m is an integerbetween 1 and 6, inclusive. In certain embodiments, n is an integerbetween 1 and 15, inclusive. In other embodiments, n is an integerbetween 1 and 10, inclusive. In yet other embodiments, n is an integerbetween 1 and 6, inclusive. In certain embodiments, n is 1. In certainembodiments, n is 2. In other embodiments, n is 3. In yet otherembodiments, n is 4. In still other embodiments, n is 6. In certainembodiments, m is 1. In other embodiments, m is 2. In yet otherembodiments, m is 3. In still other embodiments, m is 4.

In certain embodiments, B is

wherein n is an integer between 1 and 20, inclusive. In certainembodiments, n is an integer between 1 and 15, inclusive. In otherembodiments, n is an integer between 1 and 10, inclusive. In yet otherembodiments, n is an integer between 1 and 6, inclusive. In certainembodiments, n is 2. In other embodiments, n is 3. In yet otherembodiments, n is 4. In still other embodiments, n is 6.

In certain embodiments, B is

wherein n is an integer between 1 and 20, inclusive. In certainembodiments, n is an integer between 1 and 15, inclusive. In otherembodiments, n is an integer between 1 and 10, inclusive. In yet otherembodiments, n is an integer between 1 and 6, inclusive. In certainembodiments, n is 1. In certain embodiments, n is 2. In otherembodiments, n is 3. In yet other embodiments, n is 4. In still otherembodiments, n is 6.

In certain embodiments, B is

wherein n is an integer between 1 and 20, inclusive; and m is an integerbetween 1 and 6, inclusive. In certain embodiments, n is an integerbetween 1 and 15, inclusive. In other embodiments, n is an integerbetween 1 and 10, inclusive. In yet other embodiments, n is an integerbetween 1 and 6, inclusive. In certain embodiments, n is 1. In certainembodiments, n is 2. In other embodiments, n is 3. In yet otherembodiments, n is 4. In still other embodiments, n is 6. In certainembodiments, m is 1. In other embodiments, m is 2. In yet otherembodiments, m is 3. In still other embodiments, m is 4.

In certain embodiments, B is selected from the group consisting of:

In certain embodiments, the average molecular weight of the polymers ofthe present invention range from 1,000 g/mol to 50,000 g/mol, preferablyfrom 2,000 g/mol to 40,000 g/mol, more preferably from 5,000 g/mol to20,000 g/mol, and even more preferably from 10,000 g/mol to 17,000g/mol. Since the polymers of the present invention are prepared by astep polymerization, a broad, statistical distribution of chain lengthsis typically obtained. In certain embodiments, the distribution ofmolecular weights in a polymer sample is narrowed by purification andisolation steps known in the art. In other embodiments, the polymermixture may be a blend of polymers of different molecular weights.

In another embodiment, the polymer of the present invention is aco-polymer wherein one of the repeating units is a poly(β-amino ester)of the present invention. In another embodiment, the polymer of thepresent invention is a co-polymer wherein one of the repeating units isa poly(β-amino amide). Other repeating units to be used in theco-polymer include, but are not limited to, polyethylene,poly(glycolide-co-lactide) (PLGA), polyglycolic acid, polymethacrylate,etc. In certain embodiments, at least one end of the polymer is apoly(beta-amino ester), poly(beta-amino amide, or other inventivepolymer which is end-modified by the addition of a nucleophile (e.g., aamine) to a terminal acrylate unit. In certain other embodiments, atleast one end of the polymer is a poly(beta-amino ester),poly(beta-amino amide, or other inventive polymer which is end-modifiedby the addition of an electrophile to a terminal amine moiety.

Synthesis of end-modified poly(beta-amino esters)

The inventive end-modified polymers may be prepared by any method knownin the art. The polymers used as starting materials to prepareend-modified poly(beta-amino esters) or poly(beta-amino amides) areprepared from commercially available starting materials or are obtainedfrom other sources. The synthesis of poly(beta-amino esters) isdescribed in U.S. patent applications, U.S. Ser. No. 11/099,886, filedApr. 6, 2005; U.S. Ser. No. 10/446,444, filed May 28, 2003; U.S. Ser.No. 09/969,431, filed Oct. 2, 2001; U.S. Ser. No. 60/305,337, filed Jul.13, 2001; and U.S. Ser. No. 60/239,330, filed Oct. 10, 2000; each ofwhich is incorporated herein by reference.

In summary, poly(beta-amino esters) are prepared via the conjugateaddition of a diamine or primary amine to bis(acrylate esters). Thisreaction scheme is shown below:

The use of primary amines rather than diamines allows for a much widervariety of commercially available starting materials.

In preparing the polymers of the present invention, the monomers in thereaction mixture may be combined in different ratio to effect molecularweight, yield, end-termination, etc. of the resulting polymer. As wouldbe appreciated by one of skill in this art, the molecular weight of thesynthesized polymer may be determined by the reaction conditions (e.g.,temperature, starting materials, concentration, order of addition,solvent, etc.) used in the synthesis (Odian Principles of polymerization3rd Ed., New York: John Wiley & Sons, 1991; Stevens Polymer Chemistry:An Introduction 2nd Ed., New York: Oxford University Press, 1990; eachof which is incorporated herein by reference). In certain embodiments,the ratio of amine monomers to diacrylate monomers is less than 1.0. Incertain embodiments, the ratio of amine monomer to diacrylate monomer isapproximately 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, or 0.5.For example, combining the monomers at a ratio of less than 1 typicallyresults in acrylate-terminated chains, which are subsequentlyend-modified. Combining the monomers at a ratio of greater than 1typically results in amine-terminated chains, which are subsequentlyend-modified. In certain embodiments, the ratio of amine monomers todiacrylate monomers is greater than 1.0. In certain embodiments, theratio of amine monomer to diacrylate monomer is approximately 1.05, 1.1,1.15, 1.2, 1.25, 1.3, 1.35, 1.4, 1.45, 1.5, 2.0, 3.0, 4.0, or 5.0.

An acrylate-terminated poly(beta-amino ester) or other polymer,optionally purified, is reacted with a nucleophile under suitableconditions to allow the nucleophile to add to the terminal acrylateunits. In certain embodiments, an excess of the nucleophile is used inthe reaction mixture. In certain embodiments, the nucleophile used inthe reaction is an amine. In certain embodiments, the nucleophile usedin the reaction is an aniline. In other embodiments, the nucleophile isa thiol. In yet other embodiments, the nucleophile is an alcohol. Incertain embodiments, the nucleophile is a phenol. Typically, thepoly(beta-amino ester) is mixed with an excess of the nucleophile to beused to modify the terminal acrylate units.

An amine-terminated poly(beta-amino ester) or other polymer, optionallypurified, is reacted with an electrophile under suitable conditions toallow the electrophile to react with the terminal amino units. Incertain embodiments, an excess of the electrophile is used in thereaction mixture. In certain embodiments, the electrophile used in thereaction is an acrylate. In certain embodiments, the electrophile usedin the reaction is an acrylamide. In other embodiments, the electrophileis an acyl moiety. In other embodiments, the electrophile is analiphatic halide (e.g., an alkyl halide). Typically, the startingpolymer is mixed with an excess of the electrophile to be used to modifythe terminal amine units.

The reaction may be run in an organic solvent or neat. Exemplary organicsolvents include acetone, ethers, benzene, THF, toluene, hexanes, DMSO,DMF, etc. Non-nucleophilic solvents are preferred. The reaction mixturemay then be heated to effect the addition of the nucleophile to theterminal units of the polymer. In certain embodiments, the reactionmixture is heated to between 30 and 150° C. The reaction is allowed toproceed from 1 hours to 48 hours; preferably, approximately 3 hours to16 hours. As would be appreciated by one of skill in this art, thereaction conditions may vary depending on the polymer being modified andthe nucleophile being used. The progress of the reaction may beoptionally monitored by TLC, HPLC, or other analytical techniquescommonly used in the art.

The resulting end-modified polymer may be purified by any techniqueknown in the art including, but not limited to, precipitation,crystallization, chromatography, etc. In a particular embodiment, thepolymer is purified through repeated precipitations in organic solvent(e.g., diethyl ether, hexane, etc.). In another embodiment, the polymeris isolated as a salt (e.g., hydrochloride, hydrobromide, hydroiodided,phosphate, acetate, fatty acid, etc.). The resulting polymer may also beused as is without further purification and isolation.

The resulting end-modified polymer may be subsequently modified byreacting the polymer with another electrophile or nucleophile. Forexample, a nucleophile-terminated polymer may be subsequently reactedwith an electrophile. Or an electrophile-terminated polymer may besubsequently reacted with a nucleophile. The process of seriallyend-modifying a polymer may be carried out any number of times (e.g., atleast 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times. The desired polymer maybe purified at the end of each step or at the end of the process. Incertain embodiments, the individual reaction are optimized to preparethe desired polymer at greater than 80%, 90%, 95%, 98%, or 99% yield.

In one embodiment, a library of different end-modified poly(beta-aminoesters), poly(beta-amino amides, or other polymers is prepared inparallel. The synthesis of a library of end-modified polymers may becarried out using any of the teachings known in the art or describedherein regarding the synthesis of end-modified polymers. In oneembodiment, a different amine and/or bis(acrylate ester) at a particularamine-to-acrylate ratio is added to each vial in a set of vials used toprepare the library or to each well in a multi-well plate (e.g., 96-wellplate). In certain embodiments, the library contains the samepoly(beta-amino ester) core with different end modifications. Suchlibraries are particularly useful in determining the effect ofend-modification. In other embodiments, the library contains differentpoly(beta-amino esters) with the same end modification. In certainembodiments, over 100 different end-modified poly(beta-amino esters) areprepared in parallel. In certain embodiments, over 500 differentend-modified poly(beta-amino esters) are prepared in parallel. Incertain embodiments, over 1000 different end-modified poly(beta-aminoesters) are prepared in parallel. In other embodiments, over 2000different end-modified poly(beta-amino esters) are prepared in parallel.In still other embodiments, over 3000 different end-modifiedpoly(beta-amino esters) are prepared in parallel. The end-modifiedpoly(beta-amino esters) of the invention may be screened or used aftersynthesis without further precipitation, purification, or isolation ofthe polymer. In certain embodiments, the end-modified poly(beta-aminoesters) are synthesized and assayed using semi-automated techniquesand/or robotic fluid handling systems.

Uses

The inventive end-modified poly(beta-amino esters), poly(beta-aminoamides), or other polymers may be used anywhere a polymer is useful. Theuse of the end-modified polymers will depend on the physical andchemical properties of the material. Chemical properties include pKa,degradation time, ionizability, hydrophobicity, hydrophilicity,reactivity, etc.

The end-modified polymers are particularly useful in the drug deliveryarts. For example, the material may be used in forming nanoparticles,microparticles, macroparticles, capsules, coatings, or larger depots ofa therapeutic agent, diagnostic agent, or prophylatic agent. In certainembodiments, the agents to be delivered is combined with an inventivepolymer, and a therapeutically effective amount of the combination isadministered to a subject (e.g., human). Any agent may be deliveredusing the inventive materials including small molecules, contrastagents, peptides, proteins, polynucleotides, DNA, RNA, RNAi, siRNA,mRNA, tRNA, microRNA, ssDNA, dsDNA, ssRNA, shRNA, metals, organometalliccompounds, vitamins, minerals, etc. The end-modified poly(beta-aminoesters) or poly(beta-amino amides end modified with an amine areparticularly useful in delivering polynucleotides. The drug deliverydevice may provide immediate release of its payload, or it may provideextended or timed-release of the payload.

In certain embodiments, the end-modified polymer is used in tissueengineering. For example, the material may be used in bone, cartilage,liver, pancreas, and muscle replacement. In certain embodiments, thecross-linked material may be used as a bone replacement. In certainembodiments, the material includes osteoblast or other bone-formingcells, and as the material is resorbed by the body, bone is formed atthe site. In certain embodiments, the material is used in cartilagereplacement and may optionally include cells that produce cartilage orgrowth factors that induce the growth of cartilage. The inventivematerials may also be used to deliver other types of cells. The cellsmay be genetically engineered cells (e.g., they may have been altered toproduce a particular protein, peptide, or polynucleotide), or the cellsmay be wild type cells. The cells may be stem cells, pluripotent cells,or fully differentiated cells. In certain embodiments, the cells aremammalian cells. In other particular embodiments, the cells are humancells. In certain embodiments, the cells are derived from the subject(i.e., the cells are autologous). In tissue engineering uses, theend-modified polymer preferably has a degradation profile that does notinterfere with the growth of the cells. These combinations may be usedin any type of surgery including orthopedic surgery, reconstructivesurgery, plastic surgery, etc. The material may include other materialssuch as nutrients, growth factors, other polymers, materials for cellattachment, etc.

The inventive end-modified polymers also have non-medical uses. Incertain embodiments, the inventive end-modified polymer is used inpreparing a plastic products. These products typically have theadvantage of being biodegradable. The materials may also be used ascoatings, for example, coatings on papers, coatings on rock, coatings ontile, coatings on wood, coatings on flooring, coatings on metal,coatings over paint, etc. In certain embodiments, the coating is a UVprotective coating. In other embodiments, the inventive materials areused in printing. The materials may be used in inks. In still otherembodiments, the material is used as an adhesive.

Kits

The invention also provides kits for use in preparing the inventiveend-modified poly(beta-amino esters) or other polymers. The kit mayinclude any or all of the following: amines, diacrylates,poly(beta-amino esters), poly(beta-amino amides), polymers,end-modifying agents, nucleophiles, electrophiles, acrylates,acrylamides, vials, solvent, buffers, multi-well plates, salts,polynucleotides, proteins, and instructions. The instructions includeways of preparing the inventive end-modified polymers with variousproperties. In certain embodiments, the kit is tailored for preparationof end-modified polymers with a desired property or for a desired use.In certain embodiments, the kit includes all the items necessary toprepare one or more end-modified polymers.

These and other aspects of the present invention will be furtherappreciated upon consideration of the following Examples, which areintended to illustrate certain particular embodiments of the inventionbut are not intended to limit its scope, as defined by the claims.

EXAMPLES Example 1 End-Modified Poly(beta-amino esters) for GeneDelivery Introduction

Incorporation of new genetic elements into cells is a promising strategyfor the treatment of many inherited and acquired genetic disorders. Inorder for gene-based therapeutics to be clinically applicable, a safeand efficient delivery system for DNA needs to be developed. Modifiedviruses are the most heavily researched agents but still suffer frommany problems that include a limited carrying capacity, the potentialfor insertional mutagenesis, and clearance by the immune system. Variouscationic polymers have been developed as an alternative since many cancondense DNA and mediate cellular uptake. An additional benefit ofsynthetic polycations is the potential to alter the structure foroptimal gene delivery while minimizing the cytotoxic effects.

The most common polycations used for gene delivery are poly-L-lysine(PLL) and polyethylenimine (PEI). High molecular weight derivatives ofboth polymers have been shown to self-assemble with plasmid DNA to formnanoparticles capable of transfecting a variety of cell types.Furthermore, functionalization of these polymers with PEG chains andtargeting ligands has allowed for cell specific delivery, which isparticularly important for cancer gene therapies. Despite theirwidespread use, both PLL and PEI have significant disadvantages that mayultimately limit their clinical utility. In particular, both polymersare known to be very cytotoxic and have relatively low transfectionefficiencies compared to viruses, especially in non-dividing cells.

Poly(β-amino ester)s are an alternative class of cationic polymers thathave been recently developed and explored as gene delivery vectors.These polymers are degradable by hydrolysis of backbone ester bonds andcontain tertiary amines to facilitate DNA binding. They are synthesizedby a simple Michael addition reaction between bi-functional amines anddiacrylates. The polymer molecular weight and chain end groups can beeasily controlled by adjusting the amine:diacrylate monomer ratio. Thispotential variability in monomer ratio, along with the commercialavailability of many amine and diacrylate monomers, has allowed for thegeneration of large, structurally diverse libraries of poly(β-aminoester)s. High throughput transfection screens have identified manypolymers that are capable of transfecting cells with much higherefficiencies than PEI, while also demonstrating less toxicity both invitro and in vivo. The polymer libraries have also been useful toelucidate structure-function relationships. These studies have shownthat high molecular weight, amine-terminated poly(β-amino ester)s withhydroxyl-functionalized side chains are highly efficient polymers forgene delivery. The most effective polymer discovered, C32+, has alsobeen used in vivo for the gene-based treatment of prostate cancer.

The continued development of poly(β-amino ester)s for gene therapy andother biomedical applications requires an effective method to chemicallymodify these materials. As with other polycations, it is necessary toincorporate additional levels of functionality such as serum stabilityand cell targeting to improve the gene delivery properties of thesepolymers. The ideal approach to poly(β-amino ester) modification wouldinvolve a chemistry that is simple, versatile, and adaptable to a highthroughput format. It is important to generate many polymericderivatives since any modification has a non-trivial affect on genedelivery. Previous studies have shown that even single carbon orfunctional group differences between polymer repeat units candrastically affect their transfection efficiencies.

As one potential method for polymer functionalization, we present here ahigh throughput approach to synthesize end-modified poly(β-amino ester)sand explore the effects of end group structure on polymer gene deliveryproperties. End-modified polymers were synthesized following a two stepprocedure in which an acrylate-terminated base polymer is first preparedby polymerization using excess diacrylate over amine monomer. In asecond stage, the base polymer was reacted with various amine reagentsto generate amine-capped polymer chains. Following this approach, wehave generated a library of end-modified C32 polymers and demonstratethat the terminal amine has a large effect on C32 transfection. Similarto polymer repeat units, end segments differing in a single additionalcarbon or functional group can drastically affect the polymer deliveryproperties. In addition, the terminal amine structure has a largeinfluence on cytotoxicity, physical properties, and cellular uptake ofpolymer-DNA complexes. We also show that significant improvements intransfection efficiency can be made by proper end-modification of otherbase polymers that were previously found to be less effective than C32+.These results indicate that end-modification is a useful strategy tofunctionalize poly(β-amino ester)s and improve their gene deliveryperformance.

Results and Discussion

Polymer Synthesis. We developed a two-step approach to synthesizeend-modified poly(β-amino ester)s. The reactions are illustrated inFIGS. 1A and 1B for the generation of end-modified C32 polymers. In thefirst step, acrylate-terminated polymer was prepared by mixing acrylateand amine monomers in a 1.2:1.0 molar ratio, as shown previously (FIG.1A). This ratio was selected since C32+ and many other top performing,amine-terminated polymers are made at the inverse ratio using excessamine. We hypothesized then that the exact opposite ratio may be optimalso that the relative number of interior to terminal units isapproximately preserved, with the end-capping step causing a very smallchange to this balance. Since the terminal amine is very small relativeto the polymer chain, the diacrylate:amine ratio selected also controlsthe final molecular weight. For many poly(β-amino ester)s, molecularweights greater than 10 kDa are usually most effective and can beachieved using a 1.2:1.0 molar monomer ratio. For the C32-acrylatepolymer (C32-Ac), the weight-average molecular weight is approximately8,800 Da, relative to polystyrene standards, with a 1.9 polydispersityindex. Assuming that each amine-acrylate combination designates a unit,either a repeat unit in the backbone or terminal unit, then 2 of the 16units (12.5%) in an average length chain are terminal units. Thisimplies that the ends make a non-negligible contribution to the size andfunctionality of the polymer.

In the second step, acrylate-terminated polymers are reacted withvarious amine molecules to generate amine-capped polymer chains. In thisway, the chain ends contain amine functionalities different than thosepresent in the interior of the polymer. The capping reaction is shown inFIG. 1B for an arbitrary primary amine molecule, which results in anamine-capped polymer containing secondary amines at the chain endpoints. Secondary amine molecules can also be used but result intertiary amine groups at the polymer ends. The 41 different aminemolecules used for this secondary capping step are shown in FIG. 1C.These compounds were selected on the basis of their DMSO solubility andbiocompatibility. In addition, many of these molecules have provenuseful in the synthesis of poly(β-amino ester)s with high transfectionefficiencies.

The end-capping reaction occurs via an amine-acrylate Michael addition,identical to that used in the polymerization. Since the acrylatefunctionality has no detectable reactivity towards hydroxyls, ethers,tertiary amines, amides, aromatics, and some types of heterocycles, allof these functionalities can be incorporated at the chain ends using theappropriate amine reagents. The reaction is carried out by mixing aconcentrated polymer solution with an excess of amine in DMSO at roomtemperature. The conditions have been optimized with excess amine tofully end-cap all chains without causing any detectable crosslinking oraminolysis of backbone ester bonds, as determined by ¹H NMR and GPCanalysis. In addition, end-modified polymers can be directly tested fortransfection efficiency without prior purification since the DMSO andexcess amine were determined to be non-toxic (data not shown).Therefore, this chemistry permits many polymers with structurallydiverse end functionalities to be synthesized and screened in parallel.We show here that such a synthetic method is useful to assess end aminestructure-function relationships and improve the gene deliveryproperties of poly(β-amino ester)s.

In much of the analysis presented here, we use C32 as a base polymer toexamine the effects of the end amine structure on polymer transfectionand also explore structure-function relationships. This polymer wasidentified from previous studies to be the most efficient poly(β-aminoester) for gene delivery. In principle, other base polymers,diacrylate:amine ratios, and amine capping agents may be used and couldgenerate more effective polymers for gene delivery. To demonstrate thispotential, two additional amine-capped poly(b-amino ester)s, D60 andC20, were prepared and assayed for DNA transfection. The structures ofthese acrylate-terminated base polymers are shown in FIG. 1D. Also shownis the structure of acrylate-terminated AA28 polymer, which was alsocapped with several amines and used to demonstrate the ability todeliver siRNA for gene silencing.

COS-7 Transfections. The DNA delivery efficiency of end-modifiedpoly(β-amino ester)s was evaluated using a high throughput assay.Concentrated polymer solutions in DMSO were diluted in sodium acetatebuffer and complexed with plasmid DNA to form polymer-DNA nanoparticles.A range of polymer:DNA weight ratios was tested for each polymer sincethis parameter is known to have a critical effect on polycation-mediatedtransfection. Nanoparticles were then diluted into cell culture mediaand incubated on COS-7 cells. Unlike previous studies on poly(β-aminoester)s, the diluting media contained 10% serum to account for theeffect of extracellular proteins on polymer transfection.

The transfection efficiencies of amine-terminated C32 polymers are shownin FIG. 2. The average luciferase expression levels, measured inrelative light units (RLUs), are given for each polymer at fivedifferent polymer:DNA ratios. Also included is the transfection data for25 kDa branched PEI, one of the most efficient commercially availablepolycations, and C32+, the best performing poly(β-amino ester)synthesized to date. The unmodified, acrylate-terminated C32 polymer isalso shown on the far right and demonstrates weak activity that istypical of most acrylate-terminated poly(β-amino ester)s. Whileamine-capping reactions of this polymer where verified by ¹H NMRanalysis, the data in FIG. 3 provides functional confirmation by thelarge increase in transfection between acrylate- and amine-terminatedpolymers. An overall inspection of the data reveals that the structureof the terminal amine has a dramatic effect on the C32 transfectionefficiency. In general, polymers capped with hydrophilic amine endgroups containing hydroxyls or additional amines proved most effective.In contrast, chain termination with more hydrophobic amines containingalkyl chains or aromatic rings led to much lower transfection activity.

Perhaps the most important result is that very subtle structuraldifferences in just the terminal amine can have a large effect onpolymer transfection efficiency. This is most evident by comparing theC32-36 and C32-52 polymers. The C32-52 polymer, which contains asix-carbon alkyl chain extending from the terminal secondary amine, hasa maximum transfection only twice that of naked DNA. In contrast, theC32-36 polymer is 34-fold more effective than C32-52, but only differsin a single hydroxyl group on carbon-6 at the chain end. In fact, theC32-36 polymer is half as effective as C32+, demonstrating that a singlefunctional group, in this case a terminal hydroxyl, can significantlyalter the polymer delivery properties. A similar effect can be seenbetween the C32-95 and C32-110 polymers, which consist of terminaldecylamines containing either a hydroxyl group or primary amine oncarbon-10, respectively. In this case, substituting the terminalhydroxyl for an amine improves the transfection performance by over oneorder-of-magnitude. This same substitution pattern also changes theoptimal polymer:DNA ratio. Comparing two highly efficient polymers,C32-122 and C32-124, the former displays very high RLU output at a 20:1ratio, whereas the latter requires 5-fold more polymer (i.e., a 100:1ratio) to achieve the same effect. A similar trend is also seen betweenthe C32-36 and C32-106 polymers. Therefore, amine capping molecules withhydroxyls and primary amines are most effective, with the latter beingoptimal at 5-fold lower polymer:DNA ratios in general.

Polymers terminated with primary diamine molecules had the highesttransfection efficiency, as determined by both highest RLU output andlowest optimal polymer:DNA ratio. Specifically, the C32-102 polymer hada very similar transfection profile to C32+, with a maximum occurring atthe highest polymer:DNA ratio of 100:1, but had an overall 30% higherRLU output. This demonstrates that a simple modification at the chainends can significantly improve the delivery performance. Primary diaminecapping also lowered the optimal polymer:DNA ratio substantially in manycases. Seven primary amine-terminated polymers had optimal polymer:DNAratios of 20:1 while one polymer, C32-110, had a maximum RLU at a 10:1ratio. The transfection profile at the 20:1 ratio for diamine cappedpolymers, C32-102 through C32-111, appears to be a skewed bell-shapedcurve with a maximum occurring at the C32-108 polymer. This indicatesthat larger alkyl chains bridging the diamine functionalities aregenerally more effective than their short-chain counterparts, with anoptimum of eight carbons. The C32-108 polymer had an optimaltransfection at a 20:1 ratio that is almost as high as that for C32+,which requires a 100:1 ratio. Such a significant reduction in the amountof polymer needed to mediate high levels of transfection has importantimplications for in vivo delivery, where the amount of polymer injectedneeds to be limited to minimize toxic side effects.

Cytotoxicity. Many polycations have been shown to elicit considerablecell toxicity that may limit their utility as gene delivery vectors. Thebiocompatibility of cationic polymers is determined by a number offactors that include molecular weight, charge density, type of amines,polymer structure (linear, branched, dentritic), and chain flexibility.In general, high molecular weight polymers with a high density ofprimary and/or secondary amines usually result in substantiallycytotoxicity. PEI and PLL are examples of such polymers and bring aboutsignificant cell damage by compromising the cell membrane, as determinedby the cytosolic release of lactate dehydrogenase following exposure.Several poly(β-amino ester)s have shown considerably less toxicity thanPEI, presumably due to their lower molecular weights and the lack ofprimary and secondary amines. We suspected that the reduced transfectionof primary amine end-modified polymers at high polymer:DNA ratios may bedue to increased cytotoxic activity.

The cytotoxicity of end-modified poly(β-amino ester)s was evaluatedusing the MTT assay. This colorimetric test is based on the abilitymitochondrial reductase enzymes in viable cells to reduce3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to apurple formazan. New polycationic materials for biomedical applicationsare frequently tested for their effects on cellular proliferation usingthis assay. Toxicity of end-modified polymers was assessed by performingthe same transfection experiment, but assaying for metabolic activityinstead of luciferase expression. All polymers were tested at thehighest 100:1 polymer:DNA weight ratio, which corresponds to anapproximate 400 ug/ml concentration of polymer on the cells. Toxicityanalysis at such a high polymer concentration may explain thedifferences in polymer transfection at high polymer:DNA ratios, andsimultaneously assess polymer biocompatibility under very aggressiveconditions that may be important for their future use and development.

The cytotoxicity levels of end-modified C32 polymers are shown in FIG.3. The percentage of viable cells is displayed as a function of theamine-terminated polymer. Positive and negative control conditions areshown to the far right for PEI and naked DNA. At such high polymerconcentrations, PEI is known to be very cytotoxic and is reflected bythe low 3% cell viability. In contrast, both C32+ and theacrylate-terminated C32 polymer (C32-Ac) show no significant affects onthe growth and metabolism of COS-7 cells. This result for C32-Ac issomewhat surprising though since other acrylate-terminated poly(b-aminoester)s have shown considerable toxicity at this concentration.

The majority of end-modified C32 polymers show good biocompatibility.This is especially true of all polymers capped with mono-primary aminereagents, regardless of the functional groups extending from the amine.Aromatic, alkyl, hydroxyl, secondary and tertiary amines, and imidazolefunctionalities at the chain end points do not appear to invoke anyadverse effects. Therefore, elevated cytotoxic effects do notsufficiently explain the low transfection ability of polymers terminatedwith the more hydrophobic amines. In contrast to the polymers cappedwith mono-amines, polymers terminated with di-primary amine moleculescompromise cell viability to varying extents. While the increased chargeis a determining factor, the overall toxicity is also strongly dependenton the hydrophobicity of the end group. In general, increasing the sizeof the alkyl chain bridging the amine groups increases the toxicity, asis evident by comparing the C32-102 through C32-109 polymers.Furthermore, C32-121, a polymer containing a terminal polyethyleneglycolamine with an eight atom spacer between amine groups, is much less toxicthan the corresponding alkyl derivative, C32-108. This indicates thatboth the spacing between amines, and the degree of hydrophobicity in theterminal amine spacer, are important determinants of end-amine toxicity.

These significant cytotoxic effects, in large part, explain thedecreasing transfection ability of most primary diamine capped polymersat the higher polymer:DNA ratios. The additional charge, in conjunctionwith increased hydrophobicity, may be particularly damaging to the cellmembranes since both properties are known to disrupt lipid bilayers.

DNA Binding. An important requirement for cationic transfection agentsis the ability to bind and condense plasmid DNA for cell entry. Ingeneral, higher molecular weight polymers with increased cationic chargedensity display stronger DNA binding at low polymer:DNA ratios. Whilestrong electrostatic interactions are important to effectively condenseand deliver the DNA, the polymer must possess a mechanism to unbind fromthe DNA once inside the nucleus. For this reason, the poly(b-aminoester)s may be particularly advantageous since they undergo hydrolysiswith short half-lives, which may aid in DNA packaging.

The binding and condensation of DNA by polycations is often monitoredusing an agarose gel electrophoresis assay. This assay can be used toadequately determine the minimum polymer:DNA ratio required for plasmidcondensation but does not provide any information on the strength ofthis interaction. In contrast, dye binding assays provide a quantitativemeasure of the polymer-DNA binding event. As a result, we utilized aPicoGreen dye penetration assay to determine the strength and degree ofDNA binding by the end-modified poly(β-amino ester)s. In this assay,polymer-DNA complexes are formed in a manner similar to theirpreparation for transfection experiments. The complexes are then mixedwith a PicoGreen dye solution, diluted into cell culture media, and thesolution fluorescence is measured. The dye exhibits fluorescence onlywhen it intercalates between the DNA base pairs. High fluorescence istypically seen with free plasmid, but significant reductions can occurfor polymer-DNA complexes in which the DNA is partially shielded fromdye penetration. The magnitude of this fluorescence reduction relativeto free DNA correlates to the strength of the polymer-DNA interaction.

The DNA binding levels for each end-modified polymer are shown in FIG.4. Fluorescence measurements relative to free DNA are given at theoptimal transfecting polymer:DNA ratio for each polymer. With theexception of mono-amine PEG terminated polymers, all end-modifiedmaterials displayed some level of DNA binding. In general, increasedcationic charge at the end groups enabled stronger polymer-DNAinteractions. This effect is most noticeable by comparing the results ofthe PEG amine-capped polymers. The mono-amine capped polymers, C32-123and C32-124, displayed no measurable binding. However, the substitutionof a single primary amine for a hydroxyl at the chain ends (C32-121 andC32-122) leads to increased polymer-DNA binding and less dyepenetration. This result indicates that a single functional group onlyat the very end point of the polymer can bring about large changes inpolymer function. Similar conclusions were reached when assessing theoverall transfection ability of the polymer but now are seen at just onepart of the delivery process.

Perhaps the most noticeable trend in the data is that polymersterminated with primary diamine molecules are most effective atcondensing and binding DNA. Additional secondary or tertiary amines atthe chain ends were not as effective to increase the DNA binding abilityof the polymer, possibly due to pKa differences or a more stericallycrowded environment that may prevent their electrostatic interactionwith DNA. Similar to the cytotoxicity data, more effect is seen withincreased terminal hydrophobicity in addition to the added positivecharge. In general, smaller relative fluorescence is seen as the alkylchain length is increased between terminal amine groups, as is evidentby comparing polymers C32-102 through C32-110. These results aresupported by weaker DNA binding ability of polymers terminated with themore hydrophilic primary ethyleneglycol amine polymers (C32-121 andC32-122).

Particle Sizing. Simple electrostatic interactions between thepolycation and the negatively charged DNA can often lead to theirspontaneous self-assembly into cationic polymer-DNA nanoparticles. Thephysical properties of these complexes are particularly important fortheir subsequent uptake into cells. Complexes with a positive surfacecharge and a diameter less than 200 nm are usually sufficient forcellular endocytosis. These properties are dependent upon a number ofpolymer characteristics and the polymer-DNA mixing technique. Since theterminal amine has demonstrated significant effects on the DNA bindingability of polymers, it should also affect the physical properties ofthe polymer-DNA complexes.

The effective diameter of complexes formed between end-modifiedpoly(β-amino ester)s and plasmid DNA were measured using dynamic lightscattering. Polymer-DNA complexes were formed at the optimaltransfecting polymer:DNA ratio for each polycation and then diluted intocell culture media prior to each measurement. Concentrations, solutioncompositions, and polymer-DNA complexing procedures in each step wereidentical to those used in the transfection assay. In this way, thenanoparticle physical properties measured in this experiment reflect theactual particle properties in the transfection screen.

Average diameters of the polymer-DNA complexes are presented in FIG. 5for each end-modified C32 polymer. The average diameter varied between85 to 220 nm, demonstrating the crucial effects of terminal aminestructure on the physical properties of polymer-DNA complexes. Alsoshown on the far right is the average diameter of the C32+ complexes,which is determined to be 152 nm. In a previous study, C32+ complexeswere diluted into HEPES buffer and subsequently measured to be 79 nm indiameter. This difference in particle size illustrates the large effectof serum proteins to disrupt or interact with cationic polymer-DNAcomplexes. Increases in polymer-DNA complex size in the presence ofserum have been seen in studies with other polycations such as PLL andPEI, and is a well known effect on polymer-DNA properties. Importantly,the C32+ complex diameter is still below the threshold for endocytosisand maintains high transfection levels.

All end-modified polymers formed complexes with effective diameters in asuitable range for cellular uptake. Only two polymers, C32-101 andC32-121, formed complexes with diameters slightly above 200 nm. Theformer material consists of highly charged chain end groups whereas thelatter contains a short PEG diamine at the chain end points. In general,the PEG terminated polymers (C32-121 to C32-124) formed larger complexeswith diameters between 150 to 220 nm. Despite the large size and weakDNA binding of these polycations, they can still deliver DNA withrelatively high efficiencies. This effect is also true of most polymersterminated with mono-primary amine molecules. These polymers, shown onthe left side of FIG. 5, mostly result in complexes with diametersgreater than 150 nm and lower DNA binding ability than primary diaminecapped polymers. Although a general conclusion cannot be made, it isinteresting to note that some polymers with very low transfectionefficiencies (e.g., C32-117, -52, -101) also form relatively largecomplexes, suggesting that their physical properties may not beconducive to uptake.

Similar to the DNA binding data, particle sizing appears to be morefavorable for polymers capped with primary diamine molecules. For almostall of these polymers, their complexes with DNA have diameters between85-130 nm. The more hydrophilic PEG diamines, C32-121 and C32-122, arethe exception, illustrating the importance of a hydrophobic alkyl chainspace between amines at the terminus. Although the trend is not aspronounced as that for the DNA binding, it appears that the sizing issomewhat improved by increasing the alkyl chain length. This isespecially true at the long chain lengths where C32-109 and C32-110 formthe smallest complexes that have diameters less than 100 nm. Thesepolymers assemble into smaller complexes with DNA compared to thoseterminated with additional secondary and/or tertiary amines, againillustrating the benefits of primary amines at the chain ends over theseother amine functionalities. Consequently, it appears that polymersterminated with alkyl primary diamine molecules have the strongest DNAbinding characteristics and assemble into the smallest polymer-DNAcomplexes.

DNA Uptake. Differences in the physical properties of polymer-DNAcomplexes can naturally lead to differences in the rates and levels atwhich they are endocytosed into cells. Previous studies withpoly(b-amino ester)s have shown that smaller complexes with highcationic surface charge are more favorable for cellular uptake. Inaddition, amine termination has been shown to promote higher cellularinternalization over the corresponding acrylate-terminated polymer. Inlight of these findings and the terminal amine affects on polymer-DNAproperties, we measured the uptake levels of end-modified C32 polymers.Although previous studies with poly(β-amino ester)s used a novelfluorescence-based technique, we choose to use a DNA extraction andRT-PCR amplification protocol to quantify the amount of endocytosed DNA.This method provides (1) high sensitivity due to the PCR amplification,(2) linearity over several orders-of-magnitude, (3) the ability toquantify DNA uptake without pre-labeling the plasmid, and (4) ahigh-throughput, 96-well plate format to simultaneously and rapidlyanalyze all polymers. For this experiment, transfections were performedfollowing the standard protocol using a β-galactosidase (β-gal) plasmid.This DNA was isolated from the cells after a four hour post-incubationperiod and amplified using RT-PCR. The total amount of β-gal DNAharvested for each sample was calculated using a standard curve andnormalized to the COS-7 genomic DNA.

The DNA uptake levels for each end-modified polymer are shown in FIG. 6.The results are expressed as the number of plasmids endocytosed pernanogram of total DNA for each polymer at its optimal transfectingpolymer:DNA ratio. Also shown is the low uptake level of free plasmidDNA, which is most likely due to its large size and high anionic chargedensity that repels the cell surface. Positive control polymers C32+ andPEI are also shown to the far right, both of which increase DNA uptakeby approximately 5-fold over the free plasmid. On the other hand,plasmid condensation with the C32-Ac polymer did not improve uptake toany measurable level, which explains the inability of this polymer tomediate transfection. The large difference between C32+ and C32-Ac, bothin terms of uptake and transfection, highlight the importance of aminetermination to improve the C32 polymer delivery properties.

The results in FIG. 6 demonstrate that the type of amine at the chainends has a considerable effect on the endocytosis of C32 polymer-DNAcomplexes. The uptake levels varied over two orders-of-magnitude amongthe end-modified polymers, with polymer C32-106 mediating the highestplasmid internalization that is 30-fold greater than free DNA. The mostobvious trend in the data is the improved uptake that occurs forpolymers with additional terminal amines. This is evident for polymerscontaining extra secondary and tertiary end amines (C32-60 throughC32-87) and mostly for those with an additional primary end amine(C32-102 through C32-122). These results suggest that conjugation oftargeting ligands to the chain ends may be a promising strategy toachieve cell specific delivery.

The differences in uptake between each polymer also explain someimportant differences in their transfection efficiencies. First, manypolymers that are poor transfection agents also displayed very lowuptake (e.g., C32-17, -52, -93, -95). This indicates that the extracharge alone at the chain end points (compared to C32-Ac) is necessarybut not sufficient to promote C32 endocytosis. Specific functionalgroups at the chain ends, such as hydroxyls and amines, have an enhancedcapacity to interact with cell surfaces and increase uptake as comparedto more hydrophobic terminal segments. For example, the transfectiondifferences between C32-36 and C32-52 are largely related to theirdifferences in uptake. This comparison demonstrates that a singlefunctional group in the polymer chain, in this case a terminal hydroxyl,can have a large effect on cell interactions and endocytosis. Extendingthe comparison further, C32-106 differs from these two materials by aterminal primary amine. The results show that this single substitutionat the terminal amine carbon-6 can increase uptake by over 20-fold. Thiseffect is even more surprising considering that the polymer:DNA ratioused for C32-106 is 20:1, 5-fold less than that used for C32-36 andC32-52. In general, the increased uptake by the polymers capped withprimary diamines largely explains their increased effectiveness atreduced polymer:DNA ratios. The overall transfection levels may not besubstantially improved over C32+ and other non-primary amine polymersbecause (1) the transfection levels are already very high and could beclose to a saturation limit, and (2) the terminal functionalities mayhave important effects on other downstream gene delivery barriers suchas endosomal escape, cytosolic trafficking, or nuclear import.

Transfections of other Poly(β-amino ester)s. Terminal aminemodifications to the C32 polymer are shown here to have a large effecton several gene delivery properties. In particular, differences in theend amine structure have resulted in significantly improved DNA binding,the formation of much smaller polymer-DNA complexes, enhanced cellularendocytosis of these complexes, and increased transfection efficiencies,especially at the lower polymer:DNA ratios. Since the terminal amine canaffect and improve the C32 polymer performance, such modifications mayalso alter the transfection profiles and gene delivery properties ofother poly(P-amino ester)s. Furthermore, given that the terminal aminehas a large affect on cellular uptake, simple amine-capping may be aneffective means to promote cell specific delivery.

To assess the combined effects of internal polymer structure and aminetermination on transfection, we synthesized and end-capped twoadditional poly(P-amino ester)s, D60 and C20. The former is an effectivegene delivery polymer with a structure very different from that of C32.Conversely, the C20 polymer is a very poor transfection agent but isstructurally very similar to C32, differing only in the length of thealcohol side chain. These polymers were end-capped with the primarydiamine molecules that produced the most effective C32 modifications interms of both overall transfection and lower optimal polymer:DNA ratio.Transfection screens of all three polymer types (C32, D60, and C20) wereperformed on COS-7 and HepG2 cell lines. The latter was included because(1) it is a human cancer cell line frequently used to test new polymersfor gene delivery, (2) transfection of liver cells has therapeuticrelevance, and (3) it provides a second cell line to evaluate potentialtargeting effects of each polymer. Lastly, all transfections werecarried out at a 20:1 polymer:DNA weight ratio.

The transfection efficiencies for the end-modified C32, D60, and C20polymers are shown in FIG. 7. Measured RLUs are given for each polymerfor both the COS-7 (blue bars) and HepG2 (red bars) cell lines. First,it can be seen that the C32 polymers are able to transfect both cellslines, indicating that these materials may be effective delivery systemsfor a variety of cells. In general, C32 polymers terminated with primaryalkyl diamines (C32-103 through C32-108) were more effective than thosewith PEG spacers (C32-121 and -122), indicating that a degree ofhydrophobicity at the chain ends is preferential for these polymers. Forboth cell types it appears that at least a three carbon spacer betweenterminal amines is necessary to obtain effective gene delivery with C32polymers at the 20:1 ratio. The C32-103 efficiency is 130- and 300-foldhigher than C32-102 on the COS-7 and HepG2 cell lines, respectively.This result demonstrates that a single additional carbon at the chainends can alter the transfection levels by two orders-of-magnitude.

In addition to the C32 polymers, many of the end-modified D60 polymerswere highly effective gene delivery agents. In fact, ten of thesepolymers were more efficient at the reduced 20:1 ratio than C32+ at itsoptimum 100:1 ratio (FIG. 2). The best performing polymer, D60-105, hasa transfection efficiency almost 3-fold higher than the optimal C32+formulation. Unlike the end-modified C32 polymers, highly efficient D60polymers were formed with both alkyl and PEG terminal diamines. Theseresults indicate that it is necessary to concurrently optimize both theinterior sequence and end-amine structure to arrive at the mostefficient poly(β-amino ester).

In comparison to the C32 and D60 polymers, all of the C20 modificationswere much less efficient. Nevertheless, the C20 gene delivery efficiencycould be remarkably improved by proper end-functionalization. The mosteffective modified polymer, C20-108, was over two orders-of-magnitudemore efficient than C20-122. The C20-108 efficiency was still 3- to4-fold less than the optimal C32+ transfection level, but was nevertested at higher ratios where it may have better performance.Regardless, the conversion of a completely ineffective polymer into amaterial with reasonable gene delivery capabilities by end-modificationis an important result for the future development of poly(β-aminoester)s.

Some differences in polymer transfection could be seen between the COS-7and HepG2 cell lines. The most significant difference occurred for theC20-108 polymer, which was two orders-of-magnitude more effective inCOS-7 cells over the HepG2 cells. A similar but less dramatic effect wasseen with the C20-107 polymer, suggesting that C20 termination with longalkyl diamines may be a possible means to target fibroblasts. For allother polymers, including the C32 and D60, most transfection differencesbetween the cell lines were within an order-of-magnitude for eachpolymer. The inability to achieve a high level of cell specific deliveryis not surprising given that none of these end amines or polymersequences have an obvious mechanism to preferentially bind to a givencell type.

siRNA Delivery. In addition to DNA delivery, some poly(β-amino ester)shave shown the ability to deliver siRNA to down-regulate proteinexpression (unpublished data). Initial experiments with a previouspolymer library have specifically identified polymer AA28 as a promisingcandidate for further development. As a result, we synthesized anacrylate-terminated AA28 base polymer (FIG. 1D) and explored the effectsof amine end-capping on AA28 siRNA delivery. A select group of primarydiamine capping reagents was used along with the highly charged 101compound. Four additional capping reagents were included that containmultiple amines (125 through 128) to assess the effects of highlycharged ends on siRNA-mediated knockdown. As a model system, wedelivered firefly-luciferase siRNA to a HeLa cell line that stablyexpresses both firefly and renilla luciferase proteins. The decrease infirefly levels was used to quantify knockdown while any decreases inrenilla levels were used to measure and correct for cytotoxic effects.

The percent knockdown of firefly luciferase for each end-modified AA28polymer at its optimal polymer:siRNA ratio is shown in FIG. 8. Similarto the DNA delivery experiments, the end-amine structure of the polymerhas a large effect on its siRNA delivery efficiency. The most effectivepolymer discovered, AA28-126, can mediate 75% knockdown of the fireflyluciferase level. This efficiency is equal to that seen withLipofectamine, one of the most effective cationic lipid formulations forsiRNA delivery. Interestingly, the AA28-126 polymer derivative containsthe most cationic end group. In fact, the percent knockdown appears toincrease as the charge density is increased at the chain ends,indicating that this property may be generally important for siRNAdelivery with poly(β-amino ester)s.

Several effective end-modified polymers have been discovered for siRNAdelivery. With the large pool of base polymers and the availability ofmany amine molecules, a wide array of structurally diverse poly(β-aminoester)s can be prepared using the end-modification and screeningstrategy. Since the test of AA28 end capping produced several stronghits, a much larger library of materials would lead to theidentification of many poly(β-amino ester)s capable of high DNA andsiRNA delivery efficiencies.

Experimentals

Materials. Polyethylenimine (water free, M_(w)˜25 kDa, M_(n)˜10 kDa),3-amino-1-propanol (99%), N,N′-dimethylethylenediamine (99%), andanhydrous THF were purchased from Sigma-Aldrich (St. Louis, Mo.). A 25mM sodium acetate buffer solution pH 5.2 (NaAc buffer) was prepared bydiluting a 3 M stock (Sigma-Aldrich). 1,4-Butanediol diacrylate (99+%)and 5-amino-1-pentanol (97%) were from Alfa Aesar (Ward Hill, Mass.);Ethoxylated (2) bisphenol A diacrylate was from Scientific PolymerProducts, Inc. (Ontario, N.Y.); Amine capping reagents were purchasedfrom Sigma-Aldrich, Alfa Aesar, Acros Organics/Fisher Scientific(Pittsburgh, Pa.), TCI America (Portland, Oreg.), Molecular Biosciences(Boulder, Colo.), and Toronto Research Chemicals (Ontario, Canada). Allchemicals were used as received without any further purification.PicoGreen and Redi-plate 96 PicoGreen dsDNA Quantification Kit werepurchased from Molecular Probes (Eugene, Oreg.). pCMV-Luc plasmid DNAstock solution (1 mg/ml in water) was obtained from ElimBiopharmaceuticals (Hayward, Calif.). gWIZ-β-gal plasmid DNA stocksolution (5 mg/ml) was obtained from Aldevron (Fargo, N. Dak.). The MTTCell Proliferation Assay, Bright Glo™ Luciferase Assay Kits, and DualGlo™ Luciferase Assay Kits were purchased from Promega Corporation(Madison, Wis.). White and black polystyrene tissue culture treated96-well plates and half area polystyrene 96-well plates were obtainedfrom Corning Costar. Clear polystyrene tissue culture treated 96-wellplates were obtained from Becton Dickinson (Bedford, Mass.).Polypropylene 96-well deep-well plates were purchased fromSigma-Aldrich.

Cell Culture. COS-7 cells were obtained from ATCC (Manassas, Va.) andmaintained in phenol red-free DMEM supplemented with 10% fetal bovineserum and 100 units/ml of penicillin/streptomycin. HepG2 cells wereobtained from ATCC and grown in MEM supplemented with 10% fetal bovineserum, 2 mM L-glutamine, 0.1 mM non-essential amino acids, 100 units/mlof penicillin/streptomycin, and 1 mM sodium pyruvate. Luciferaseexpressing HeLa cells were maintained in DMEM supplemented with 10%fetal bovine serum, 100 units/ml of penicillin/streptomycin, 500 ug/mlzeocin (Sigma-Aldrich), and 0.5 ug/ml puromycin. All cell culturereagents were purchased from Invitrogen Corporation (Carlsbad, Calif.)unless otherwise noted. All cell lines were grown at 37° C., 5% CO₂atmosphere.

Methods. ¹H NMR was conducted on a Varian Unity spectrometer (300 MHz).

Synthesis of Acrylate-Terminated Poly(β-amino ester)s. Allacrylate-terminated polymers were synthesized by mixing the appropriatemonomers in a 1.2:1.0 molar ratio of diacrylate:amine. C32-Ac wasprepared by mixing 793 mg of 1,4-butanediol diacrylate (4 mmol) with 344mg of 5-amino-1-pentanol (3.3 mmol). D60-Ac was prepared by mixing 1443mg of ethoxylated (2) bisphenol A diacrylate (3.4 mmol), 250 mg ofN,N′-dimethylethylenediamine (2.8 mmol), and 1 ml of DMSO. C20-Ac wasprepared by mixing 793 mg of 1,4-Butanediol diacrylate (4 mmol) with 250mg of 3-amino-1-propanol (3.3 mmol). Polymerizations were performed inTeflon-lined screw cap vials under magnetic stirring at 90° C. for 24hours. ¹H NMR of C32-Ac (d₆-DMSO): δ (ppm) 1.2-1.4 (m,—NCH₂(CH₂)₃CH₂OH), 1.6 (bs —N(CH₂)₂COOCH₂CH₂—), 2.4 (m, —COOCH₂CH₂N— and—NCH₂(CH₂)₄OH), 2.6 (m, —COOCH₂CH₂N—), 3.4 (bs, —N(CH₂)₄—CH₂OH), 4.0(bs, —N(CH₂)₂COOCH₂CH₂—), 4.1 (t, CH₂CHCOOCH₂CH₂—), 4.4 (bs,—N(CH₂)₅OH), 5.9 (d, CH₂CHCOOCH₂CH₂—), 6.2 (m, CH₂CHCOOCH₂CH₂—), 6.3 (d,CH₂CHCOOCH₂CH₂—).

Synthesis of Amine-Capped Poly(β-amino ester)s. Acrylate-terminatedpolymers were dissolved in DMSO at 31.13% wt/wt. Amine capping reagentswere dissolved in DMSO at 0.25 M. End chain capping reactions wereperformed by mixing 321 mg of polymer/DMSO solution with 800 μl of aminesolution. Reactions were performed in eppendorf tubes with constantagitation for 24 hours. Polymers were stored at −20 deg C. until usedfor each experiment. ¹H NMR of C32-Ac capped with 5-amino-1-pentanol(C32-32) (d₆-DMSO): δ (ppm) 1.2-1.4 (m, —NCH₂(CH₂)₃CH₂OH), 1.6 (bs—N(CH₂)₂COOCH₂CH₂—), 2.4 (m, —COOCH₂CH₂N— and —NCH₂(CH₂)₄OH), 2.6 (m,—COOCH₂CH₂N—), 3.4 (m, —N(CH₂)₄—CH₂OH), 4.0 (bs, —N(CH₂)₂COOCH₂CH₂—).

Polymer Transfections. COS-7 cells (15,000 cells/well) or HepG2 cells(5,000 cells/well) were plated in opaque 96-well plates and allowed toadhere overnight. Polymers at 100 mg/ml in DMSO were diluted accordinglyinto NaAc buffer to concentrations that yield the different polymer:DNAweight ratios. One hundred microliters of diluted polymer solution wasmixed vigorously with 100 μl of DNA (60 μg/ml in NaAc buffer) in a96-well polystyrene plate. The solutions were left undisturbed for 5minutes after which time 120 μl of each was added to 800 μl of cellculture media in a deep-well polypropylene plate. The media over thecells was then removed with a 12-channel aspirator wand and followed bythe addition of 150 μl/well of polymer-DNA complex solution. Complexeswere incubated over the cells for one hour after which time they wereaspirated off and replaced with 105 μl/well of fresh cell culture media.Cells were allowed to grow for three days at 37° C., 5% CO₂ and thenanalyzed for luciferase protein expression.

Luciferase expression was analyzed using Bright-Glom assays kits.Briefly, 100 μl/well of Bright-Glo solution was added to the cellplates. The plates were gently agitated to promote mixing for 2 minutes.Luminescence was then measured on a Perkin Elmer Victor 3 plateluminometer using a 1% neutral density filter and a one second per wellcounting time.

Measurements of Polymer Cytotoxicity. Cytotoxicity measurements ofpolymer-DNA complexes were performed essentially as described for thetransfection experiments except that cellular metabolic activity wasmeasured instead of Luciferase protein expression. One day after thetransfection, MTT reagent was added to the cell plates at 10 μl/well.The plates were incubated at 37° C. for 2 hours. Detergent reagent wasthen added at 100 μl/well and the cell plates were left in the dark atroom temperature for 4 hours. Optical absorbance was measured at 570 nmusing a Molecular Devices SPECTRAmax PLUS384 absorbance plate reader andconverted to percent cell viability relative to untreated cells.

Polymer-DNA Binding Assay with PicoGreen. Polymer solutions at 100 mg/mlin DMSO were diluted into NaAc buffer to a final concentration of 6mg/ml. In a half area 96-well plate, 50 μl/well of diluted polymer wasadded to 50 μl/well of DNA (60 μg/ml in NaAc buffer). The solutions weremixed vigorously and allowed to sit undisturbed for 5 minutes to allowfor polymer-DNA complexation. After this time, 100 μl/well of PicoGreensolution was added. PicoGreen working solution was prepared by diluting80 μl of the purchased stock into 15.2 ml NaAc buffer. After 5 minutes,30 μl/well of polymer-DNA-PicoGreen solution was added to 200 μl/well ofDMEM media in black 96-well polystyrene plates. The plate fluorescencewas then measured on a Perkin Elmer Victor 3 plate reader using a FITCfilter set (excitation 485 nm, emission 535 nm). The relativefluorescence (RF) was calculated using the following relationship:

RF=(F _(sample) −F _(blank))/(F _(DNA)−F_(blank))

where F_(sample) is the fluorescence of the polymer-DNA-PicoGreensample, F_(blank) is the fluorescence of a sample with no polymer or DNA(only PicoGreen), and F_(DNA) is the fluorescence of DNA-PicoGreen (nopolymer).

Polymer-DNA Complex Size. Polymer solutions at 100 mg/ml in DMSO werediluted into NaAc buffer the appropriate concentration. Concentrationswere adjusted for each polymer so that the final polymer:DNA ratio wasthe same that produced the highest transfection. To prepare polymer:DNAcomplexes, 100 μl of diluted polymer was added to 100 μl of DNA (60μg/ml in NaAc) and pipetted vigorously. Complexation was allowed toproceed undisturbed for 5 minutes after which time 150 μl of the samplewas diluted into 1.8 ml of DMEM media. Polymer:DNA complex size wasmeasured on a ZetaPALS dynamic light scattering detector (BrookhavenInstruments Corporation, Holtsville, N.Y.; 15 mW laser; 676 nm incidentbeam, 90° scattering angle). Effective particle diameters werecalculated from the autocorrelation function using the MAS option of theBIC particle sizing software assuming a log normal distribution. Thesolution viscosity and refractive index were assumed equal to pure waterat 25° C.

Cellular Uptake Assay. Uptake measurements of polymer-DNA complexes wereperformed essentially as described for the transfection experiments butusing the b-galactosidase plasmid. Instead of quantifying proteinexpression levels after three days, total cellular DNA was isolatedfours hours post-transfection using a DNeasy 96 Tissue Kit (Qiagen;Valencia, Calif.) following the manufacturer instructions. Total DNA wasquantified using a Redi-Plate 96 PicoGreen dsDNA Quantification kitfollowing the supplied instructions. The amount of 1-gal DNA deliveredwas quantified using RT-PCR with a Taqman primer and probe set specificfor the 1-gal plasmid (Applied Biosystems; Foster City, Calif.). Afteractivating the Taq enzyme at 95° C., 40 cycles of amplification wereperformed, with each cycle consisting of 95° C. for 15 seconds, 60° C.for one minute, followed by a fluorescent plate read using a Chromo4Continuous Fluorescence Detector (MJ Research; Waltham, Mass.). Plasmidcopy numbers were determined by comparing the RT-PCR cycle thresholdvalues to a plasmid standard curve and analyzed using the OpticonMonitor 3 software package (MJ Research).

siRNA Delivery. HeLa cells (15,000 cells/well) stably expressing fireflyand renilla luciferase proteins were plated in opaque 96-well plates andallowed to adhere overnight. Polymers at 100 mg/ml in DMSO were dilutedaccordingly into NaAc buffer to concentrations that yield the differentpolymer:RNA weight ratios. Twenty five microliters of diluted polymersolution was mixed vigorously with 25 μl of RNA (30 μg/ml in NaAcbuffer) in a 96-well polystyrene plate. The solutions were leftundisturbed for 20 minutes after which time 30 μl of each was added to200 μl of cell culture media in a deep-well polypropylene plate. Themedia over the cells was then removed with a 12-channel aspirator wandand followed by the addition of 150 μl/well of polymer-RNA complexsolution. Complexes were incubated over the cells for one day (37° C.,5% CO₂) after which time they were aspirated off and assayed forluciferase expression using the Dual Glo™ Luciferase Assay following themanufacturer instructions. Luminescence was measured on a Perkin ElmerVictor 3 plate luminometer using a one second per well counting time.The percent knockdown (% KD) was calculated for each polymer inquadruplicate using the following equations:

% KD=1−(F _(f))_(p) /[n*(F _(f))_(u)]

n=(F _(R))_(p)/(F _(R))_(u)

where (F_(f))_(p) is the measured firefly fluorescence of a polymersample, (F_(f))_(u) is the measured firefly fluorescence of theuntreated cells, (F_(R))_(p) is the measured renilla fluorescence of apolymer sample, and (F_(R))_(u) is the measured renilla fluorescence ofthe untreated cells.

Other Embodiments

The foregoing has been a description of certain non-limiting preferredembodiments of the invention. Those of ordinary skill in the art willappreciate that various changes and modifications to this descriptionmay be made without departing from the spirit or scope of the presentinvention, as defined in the following claims.

1. (canceled)
 2. A polymer of formula:

wherein B are linkers that may be any substituted or unsubstituted,branched or unbranched, cyclic or acyclic aliphatic or heteroaliphaticmoiety; or substituted or unsubstituted aryl or heteroaryl moieties;each of R₁, R₃, and R₄ are independently a hydrogen; halogen; branchedor unbranched, substituted or unsubstituted, cyclic or acyclicaliphatic; branched or unbranched, substituted or unsubstituted, cyclicor acyclic heteroaliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; X is O, S, NH, orNR_(X), wherein R_(X) is halogen; branched or unbranched, substituted orunsubstituted, cyclic or acyclic aliphatic; branched or unbranched,substituted or unsubstituted, cyclic or acyclic heteroaliphatic;branched or unbranched, substituted or unsubstituted, cyclic or acyclicacyl; substituted or unsubstituted aryl; or substituted or unsubstitutedheteroaryl; and n is an integer between 5 and 10,000, inclusive; or apharmaceutically acceptable salt thereof.
 3. (canceled)
 4. A polymer offormula:

wherein A and B are linkers that may be any substituted orunsubstituted, branched or unbranched, cyclic or acyclic aliphatic orheteroaliphatic moiety; or substituted or unsubstituted aryl orheteroaryl moieties; each of R₁, R₂, R₃, and R₄ are independently ahydrogen; halogen; branched or unbranched, substituted or unsubstituted,cyclic or acyclic aliphatic; branched or unbranched, substituted orunsubstituted, cyclic or acyclic heteroaliphatic; branched orunbranched, substituted or unsubstituted, cyclic or acyclic acyl;substituted or unsubstituted aryl; or substituted or unsubstitutedheteroaryl, wherein R₁ and R₂ may optionally form a cyclic structure orR₁ and R₂ may optionally form cyclic structures with A; and X is O, S,NH, or NR_(X), wherein R_(X) is halogen; branched or unbranched,substituted or unsubstituted, cyclic or acyclic aliphatic; branched orunbranched, substituted or unsubstituted, cyclic or acyclicheteroaliphatic; branched or unbranched, substituted or unsubstituted,cyclic or acyclic acyl; substituted or unsubstituted aryl; orsubstituted or unsubstituted heteroaryl; and n is an integer between 5and 10,000, inclusive; or a pharmaceutically acceptable salt thereof. 5.(canceled)
 6. A polymer of formula:

wherein A and B are linkers that may be any substituted orunsubstituted, branched or unbranched, cyclic or acyclic aliphatic orheteroaliphatic moiety; or substituted or unsubstituted heteroarylmoieties; each of R₁, R₂, R₃, and R₄ are independently a hydrogen;halogen; branched or unbranched, substituted or unsubstituted, cyclic oracyclic aliphatic; branched or unbranched, substituted or unsubstituted,cyclic or acyclic heteroaliphatic; branched or unbranched, substitutedor unsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl, wherein R₁ and R₂ mayoptionally form a cyclic structure, or R₁ and R₂ may optionally formcyclic structures with A; and X is O, S, NH, or NR_(X), wherein R_(X) ishalogen; branched or unbranched, substituted or unsubstituted, cyclic oracyclic aliphatic; branched or unbranched, substituted or unsubstituted,cyclic or acyclic heteroaliphatic; branched or unbranched, substitutedor unsubstituted, cyclic or acyclic acyl; substituted or unsubstitutedaryl; or substituted or unsubstituted heteroaryl; and n is an integerbetween 5 and 10,000, inclusive; or a pharmaceutically acceptable saltthereof.
 7. (canceled)
 8. The polymer of claim 4, wherein A is

wherein n is an integer between 1 and 20, inclusive.
 9. The polymer ofclaim 4, wherein A is

wherein n is an integer between 1 and 20, inclusive.
 10. The polymer ofclaim 4, wherein A is

wherein n is an integer between 1 and 20, inclusive; and m is an integerbetween 1 and 6, inclusive.
 11. The polymer of claim 2, wherein B is ansubstituted or unsubstituted, branched or unbranched, aliphatic orheteroaliphatic moiety.
 12. The polymer of claim 2, wherein B is

wherein n is an integer between 1 and 20, inclusive.
 13. The polymer ofclaim 2, wherein B is

wherein n is an integer between 1 and 20, inclusive.
 14. The polymer ofclaim 2, wherein B is

wherein n is an integer between 1 and 20, inclusive; and m is an integerbetween 1 and 6, inclusive.
 15. The polymer of claim 2, wherein B is:


16. (canceled)
 17. The polymer of claim 2, wherein X is NH, NR_(X), orNMe.
 18. (canceled)
 19. (canceled)
 20. The polymer of claim 2, whereinR₃ and R₄ are the same.
 21. The polymer of claim 2, wherein R₃ and R₄are different.
 22. (canceled)
 23. The polymer of claim 2, wherein R₁ ishydroxyalkyl.
 24. (canceled)
 25. The polymer of claim 2, wherein R₁ is:

26.-30. (canceled)
 31. The polymer of claim 4, wherein R₁ and R₂ are thesame.
 32. The polymer of claim 2, wherein R₃ and R₄ are

wherein m is an integer between 1 and 20, inclusive. 33.-35. (canceled)36. The polymer of claim 2, wherein R₃ and R₄ are

wherein m is an integer between 1 and 20, inclusive.
 37. (canceled) 38.(canceled)
 39. The polymer of claim 2, wherein R₃ and R₄ are

wherein n, m, and p are each independently an integer between 0 and 20,inclusive; and V is —O—, —S—, —NH—, —NR_(V)—, or C(R_(V))₂, whereinR_(V) is hydrogen, hydroxyl, C₁₋₆aliphatic, C₁₋₆heteroaliphatic,C₁₋₆alkoxy, amino, C₁₋₆alkylamino, di(C₁₋₆alkyl)amino, aryl, heteroaryl,thiol, alkylthioxy, or acyl. 40.-42. (canceled)
 43. The polymer of claim2, wherein R₃ and R₄ are selected from the group consisting of:


44. The polymer of claim 2, wherein the polymer has a molecular weightbetween 1,000 and 100,000 g/mol.
 45. (canceled)
 46. (canceled)
 47. Apharmaceutical composition comprising a polynucleotide and a polymer ofclaim
 1. 48. (canceled)
 49. (canceled)
 50. The pharmaceuticalcomposition of claim 47, wherein the polynucleotide is an siRNA. 51.-63.(canceled)
 64. A method of synthesizing an end-modified poly(β-aminoester), the method comprising steps of: providing an acrylate-terminatedpoly(beta-amino ester); providing an amine; and reacting the amine andthe acrylate-terminated poly(beta-amino ester) under suitable conditionsto form an end-modified poly(β-amino ester). 65.-87. (canceled)